Formulations and methods of use for alpha connexin c-terminal (act) peptides

ABSTRACT

This invention relates to a topical gel drug product preparation containing a composition comprising an isolated polypeptide having a carboxy-terminal amino acid sequence of an alpha connexin (ACT peptide), peptide stabilizers, excipients, buffering agents, and the like. A formulation and preparation steps are disclosed for the manufacturing of a stable, elegant, and pourable topical gel. The resulting formulation possesses long term stability suitable for aesthetic as well as therapeutic applications including the prevention of scaring and accelerated healing of wounds. Methods for treatment of chronic wounds, including chronic ulcers, are also provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of PCT Application NO.PCT/US2013/028727, filed Mar. 1, 2013, which claims priority to U.S.Provisional Patent Application No. 61/605,528, filed Mar. 1, 2012, whichis incorporated herein by reference in its entirety.

DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY

The contents of the text file submitted electronically herewith areincorporated herein by reference in their entirety: A computer readableformat copy of the Sequence Listing (filename:FIRS_(—)006_(—)01US_SeqList_ST25.txt, date recorded: Mar. 15, 2013, filesize 32 kilobytes).

TECHNICAL FIELD

The present invention relates to compositions for wound treatmentcomprising alpha connexin polypeptides and stabilizing agents. Topicalformulations containing alpha connexin polypeptides andhydroxyethylcellulose are particularly stable. These compositions may beused to treat a variety of wounds including acute surgical wounds andchronic ulcers. Methods for treatment of chronic wounds are alsoprovided.

BACKGROUND

While human tissues damaged by mechanical wounding, disease processesand other causes are capable of healing, complex tissue structure andfunction is rarely, if ever wholly restored. Instead, recovery of nearlyall tissues from injury in humans and other higher vertebrates isdominated by the formation of scar tissue. The most familiar example ofthis is the discolored and fibrotic scars that linger following thehealing of a skin cut or graze. Less well appreciated is that formationof glial scar tissue following injury to the brain or spinal chord isone of the main obstacles to restoration of neural function followingdamage to the central nervous system (Silver and Miller J H, 2004).There is currently no means of treating or preventing such scarring andpromoting the regeneration of complex tissue structure and functionfollowing injury.

Once such treatments are discovered, there is also a need to provideformulations that stabilize the active pharmaceutical ingredients (API)which they contain. While there are a myriad of potential stabilizingagents, not all of them work equally well in stabilizing specific APIs.

Venous ulcers are chronic wounds associated with long-standing venoushypertension of the lower extremity. The number of individuals affectedby these ulcers in the United States is over 600,000. These indolentwounds are a major cause of morbidity and are a major financial burden.Diabetes affects an estimated 25.8 million people (8% of the USpopulation) according to the Centers for Disease Control and Prevention.People with diabetes have 12-25% chance of developing foot ulcer intheir lifetime. Diabetic Foot Ulcers (DFUs) refer to wounds below theankle of a diabetic subject which develops due to neuropathy (sensory,motor, or autonomic deficits), ischemia or both. Foot ulcers are asubstantial cause for morbidity and can severely impair quality of life,engender high treatment costs, and serve as the most important riskfactor for lower-extremity amputation.

No single available product or procedure is adequate for the treatmentof all subjects with chronic ulcers. Treatment selection is determinedby patient tolerance, patient medical status, cost, availability, andphysician preference. Compression wraps are the Standard of Care (SoC)and are needed to assist venous return and to address the underlyingpathophysiology of venous disease and venous ulceration. Compressionbandages and stockings heal more ulcers compared with no compression,but it has not been ascertained if intermittent pneumatic compression isbeneficial compared with compression bandages or stockings. Occlusive(hydrocolloid) dressings are no more effective than simple low-adherentdressings in people treated with compression.

Despite various advances in wound treatment, the need exists foradditional effective VLU and DFU treatments that combine advanced woundcare therapeutics with SoC compression therapy for faster healing ofulcers. Rapid healing would result in fewer hospitalizations, decreasedinfection, less use of antibiotics, improved mobility, less pain, andfewer surgical procedures for subjects with VLUs or DFUs.

SUMMARY OF THE INVENTION

The present invention is directed in part to a topical gel drug productpreparation containing a composition comprising an isolated alphaconnexin polypeptide. The topical formulation may further comprisehydroxyethylcellulose gel, which stabilizes the alpha connexinpolypeptide during storage. In certain embodiments the alpha connexinpolypeptide comprises a carboxy terminal amino acid sequence of an alphaconnexin (hereinafter “ACT”). The alpha connexin polypeptides of thepresent invention may comprise or consist of the carboxy-terminal most 4to 30 contiguous amino acids of an alpha connexin protein orconservative variant thereof, wherein said at least one alpha connexinpolypeptide is linked at its amino terminus to a cellularinternalization transporter.

It is an object of the present invention to prepare a formulation of astable, elegant, and pourable topical gel carrier that contains ACT foraesthetic as well as therapeutic applications including the preventionof scaring and accelerated healing of wounds of a subject. The drugproduct may be administered to treat acute surgical wounds to reducescarring or be applied chronically to difficult-to-heal woundsincluding, but not limited to, venous leg ulcers, diabetic foot ulcers,pressure ulcers, and the like. The drug product possessesphysicochemical, biochemical, and rheological properties that enable itsability to provide a therapeutic and effective amount of ACT peptidewhen applied to all type of wounds.

In one aspect of the invention, one or more stabilizer is selected amongmany, and prepared with ACT peptide to stabilize the isolatedpolypeptide. Preferably the stabilizers are non-irritating,non-staining, and non-immunogenic. The stabilizers enable the long term(i.e., for 3 months, for 6 months, for 9 months, for 12 months, for 18months, or for 24 months) storage of the drug product under a variety oftemperature conditions (i.e., at about 5° C., at about 10° C., at about15° C., at about 20° C., at about 25° C., at about 30° C., at about 35°C., or at about 40° C.) and under a range of relative humidities (i.e.,at 0% relative humidity, at 10% relative humidity, at 20% relativehumidity, at 30% relative humidity, at 40% relative humidity, at 50%relative humidity, at 60% relative humidity, at 70% relative humidity,at 80% relative humidity, at 90% relative humidity, or at 100% relativehumidity).

In another aspect of the invention, one or more excipients are selectedfrom the group consisting of water-soluble porogens, polymers, ioniccompounds, non-ionic compounds, and solvents. In a further embodiment,excipients act as one or more of carriers, stabilizers, and gellingagents. In one embodiment, at least one excipient is prepared with ACT.Preferably the excipients are non-irritating, non-staining, andnon-immunogenic. In one embodiment the formulation comprises at leastone polymer. In a further embodiment, the formulation comprises aderivative of cellulose. In a yet further embodiment, the derivative ofcellulose is hydroxyethylcellulose.

In yet another aspect of the invention, an optimal pH range for thetopical gel is about pH 5 to about pH 7, and is maintained by theaddition of one or more buffering agents. Preferably the bufferingagents are non-irritating, non-staining, and non-immunogenic.

In yet another aspect of the invention, at least one excipient is agelling agent. In one embodiment, a gelling agent is a carrier of ACT.In some embodiments, the gelling agent also acts as a stabilizer. Insome embodiments, the gelling agent may be combined with at least one ofthe following: one or more buffering agent, and one or more additionalexcipient. In one embodiment, the gelling agent ishydroxyethylcellulose.

In some embodiments, the present invention includes a method of woundtreatment comprising administering to a subject in need thereof atopical formulation comprising at least one alpha connexin polypeptideand hydroxyethylcellulose gel. In an exemplary embodiment, the steps aredisclosed for manufacturing of the drug product.

The present invention also includes methods of treating wounds andulcers in a patient in need thereof, particularly wounds and ulcers thatare chronic in nature. The present inventors have found that thepeptides and formulations of the present invention provide fasterhealing of ulcers when combined with compression therapy than isachieved by compression therapy alone. Accordingly, the presentinvention includes methods of treating a chronic wound in a subject,comprising administering to the subject a topical formulation comprisingat least one alpha connexin polypeptide in addition to standard of carecompression therapy, wherein the chronic wound is healed at a fasterrate and/or increased frequency than achieved with standard of carecompression therapy alone.

In one aspect a method of treating a chronic wound in a subjectcomprising administering to the subject a topical formulation comprisingat least one alpha connexin polypeptide is provided. In a furtherembodiment, the formulation is administered daily or weekly or acombination thereof. In another embodiment, the formulation isadministered according to any dosing regiment that is effective in thetreatment of the chronic wound. In a further embodiment, the formulationis administered in a dosing regimen at day 0, day 3, week 1, week 2,week 3, week 4, week 5, week 6, week 7, week 8, week 9, week 10, week11, and week 12.

In one embodiment a method of treating a chronic wound in a subjectcomprising administering to the subject a topical formulation comprisingat least one alpha connexin polypeptide and hydroxyethylcellulose isprovided. In a further embodiment, the hydroxyethylcellulose is presentat a concentration of about 1.25% (w/w). In one embodiment, the at leastone isolated polypeptide comprises the carboxy terminal-most 4 to 30contiguous amino acids of an alpha Connexin, or a conservative variantthereof. In another embodiment, the alpha connexin polypeptide isconnexin 37, connexin 40, connexin 43, or connexin 45. In oneembodiment, the alpha connexin is linked at its amino terminus to acellular internalization transporter such as, for example anAntennapedia sequence, TAT, HIV-Tat, Penetratin, Antp-3A (Antp mutant),Buforin II, Transportan, MAP (model amphipathic peptide), K-FGF, Ku70,Prion, pVEC, Pep-1, SynB1, Pep-7, HN-1, BGSC(Bis-Guanidinium-Spermidine-Cholesterol, and BGTC(BisGuanidinium-Tren-Cholesterol). In one embodiment, the cellularinternalization transporter is an antennapedia sequence. In a furtherembodiment, the formulation comprises a polypeptide comprising the aminoacid sequence of SEQ ID NO: 9.

In one embodiment, the formulation for use in treatment of a chronicwound comprises at least one alpha connexin polypeptide andhydroxyethylcellulose gel. In a further embodiment, the hydroxycellulosegel is present at a concentration of about 2%, about 1.75%, about 1.5%,about 1.25%, about 1.0%, or about 0.75%. In one embodiment, thehydroxycellulose gel is present at a concentration of about 1.25% (w/w).

In one aspect, the chronic wound is an ulcer. In a further embodiment,the chronic wound is a lower extremity ulcer. In another embodiment, thechronic wound is selected from the group consisting of venous legulcers, diabetic foot ulcers, and pressure ulcers. In one embodiment, amethod for treating a chronic wound in a subject is provided, whereinthe method comprises administering to the subject a topical formulationof at least one alpha connexin polypeptide, wherein the formulation isadministered in a dosing regimen at day 0, day 3, week 1, week 2, week3, week 4, week 5, week 6, week 7, week 8, week 9, week 10, week 11, andweek 12, wherein the symptoms of the chronic wound are reduced. In afurther embodiment, the formulation does not induce excessive levels ofside effects. In another embodiment, the chronic wound is improved inthe absence of clinically significant abnormalities. In anotherembodiment, the method reduces the time to 100% wound closure, ascompared to the time to 100% wound closure when the standard of caretreatment is used. In other embodiments, the method reduces the time to100% wound closure when administered in conjunction with standard ofcare, as compared to treatment with either standard of care alone.

Standard of care treatments are known to those of skill in the art andinclude, for example, cleaning the wound, applying dressing to thewound, and applying a pressure bandage to the wound. In one embodiment,the subject treated with the methods and formulations provided hereinhas a higher percent wound closure as compared to a subject receivingstandard of care therapy, at 12 weeks, 11 weeks, 10 weeks, 9 weeks, 8weeks, 7 weeks, 6 weeks, 5 weeks, 4 weeks, 3 weeks, or 2 weeks. In oneembodiment, the percent wound closure at 12 weeks is higher in subjectstreated with the methods and formulations disclosed herein, as comparedto the percent wound closure at 12 weeks in subjects receiving standardof care treatment. In one embodiment, the method reduces the time to 50%wound closure, as compared to the time to 50% wound closure when thestandard of care treatment is used. In another embodiment, the percentwound closure at 4 weeks is higher in subjects treated with the methodsand formulations disclosed herein, as compared to the percent woundclosure at 4 weeks in subjects treated with standard of care treatment.

In another embodiment, the method results in a reduction in pain levelsin the subject. In a further embodiment, the pain level is determinedthrough patient self-assessment. In one embodiment, the method increasesthe average percent of wound closure at 12 weeks, 11 weeks, 10 weeks, 9weeks, 8 weeks, 7 weeks, 6 weeks, 5 weeks, 4 weeks, 3 weeks, or 2 weeksas compared to standard of care treatment. In one embodiment, the methodincreases the average percent of wound closure at 12 weeks. In oneembodiment, the method decreases the wound area as compared to the woundarea in subjects that are treated with standard of care therapy. In oneembodiment, the method does not induce the production of anti-alphaconnexin polypeptide antibodies in the subject.

In another embodiment, the method increases the incidence or frequencyof 100% complete wound closure compared to standard of care treatmentsfor wound healing. In another embodiment, the method is used to treat anulcer lacking sufficient wound size reduction within one, four, 12, 24,36, or more weeks of standard of care. In one embodiment, the method isused to treat an ulcer with less than 50% wound closure within one four,12, 24, 36, or more weeks of standard of care. In one embodiment, themethod is used to treat an ulcer lacking sufficient wound size reductionwithin one, four, 12, 24, 36, or more weeks of standard of care. In oneembodiment, the method is used to treat an ulcer lacking sufficientwound size reduction within one, four, 12, 24, 36, or more weeks ofstandard of care. In one embodiment, the method is used to treat apatient with an ulcer possessing wound area and duration characteristicsof a chronic wound. In one embodiment, the method is used to treat apatient with an ulcer possessing a wound with a non-healing woundtrajectory. In one embodiment, the method is used to treat an ulcerpossessing wound area and time duration characteristics of a chronicwound.

DESCRIPTION OF DRAWINGS

For a fuller understanding of the nature and advantages of the presentinvention, reference should be had to the following detailed descriptiontaken in connection with the accompanying drawings, in which:

FIG. 1 illustrates the manufacturing process flow chart.

FIG. 2 shows the subject self assessment of pain in the intent-to-treatpopulation in the Diabetic Foot Ulcer (DFU) study.

FIG. 3 shows the subject self assessment of pain in the per-protocol(PP) population in the DFU study.

FIG. 4 shows the time to 100% wound closure in the intent-to-treat (ITT)population in the DFU study.

FIG. 5 shows the time to 100% wound closure in the PP population in theDFU study.

FIG. 6 shows the time to 50% wound closure in the ITT population in theDFU study.

FIG. 7 shows the time to 50% wound closure in the PP population in theDFU study.

FIG. 8 shows the average wound closure at each visit in the ITTpopulation in the DFU study.

FIG. 9 shows the average wound closure at each visit in the PPpopulation in the DFU study.

FIG. 10 shows the Longitudinal Response Profile of the Mean PercentReduction of Wound Area (mm²) from Baseline to Week 12 by TreatmentGroup—ITT Population (N=92)

FIG. 11 Longitudinal Response Profile of the Mean Percentage Reductionof Wound Area from Baseline to Week 12 by Treatment Group—mITTPopulation (N=77)

FIG. 12 shows the Longitudinal Response Profile of the Mean PercentageReduction of Wound Area (mm²) from Baseline to Week 12 by TreatmentGroup—PP Population (N=68)

FIG. 13 shows the Kaplan-Meier Plot of Time to First 100% Wound ClosureITT Population (N=92)

FIG. 14 shows the Kaplan-Meier Plot of Time to First 100% Wound ClosureITT Population (N=92)

FIG. 15 shows Kaplan-Meier Plot of Time to First 100% Wound Closure PPPopulation (N=68)

FIG. 16 shows the Kaplan-Meier Plot of Time to First 50% Wound ClosureITT Population (N=92)

FIG. 17 shows the Kaplan-Meier Plot of Time to 50% Wound Closure-mITTPopulation (N=77)

FIG. 18 shows the Kaplan-Meier Plot of Time to First 50% Wound ClosurePP Population (N=68)

DETAILED DESCRIPTION

Before the present compositions and methods are described, it is to beunderstood that this invention is not limited to the particularprocesses, compositions, or methodologies described, as these may vary.The terminology used in the description is for the purpose of describingthe particular versions or embodiments only, and it is not intended tolimit the scope of the present invention which will be limited only bythe appended claims. Unless defined otherwise, all technical andscientific terms used herein have the same meanings as commonlyunderstood by one of ordinary skill in the art. All publicationsmentioned herein are incorporated by reference in their entirety.Nothing herein is to be construed as an admission that the invention isnot entitled to antedate such disclosure by virtue of prior invention.

It must also be noted that as used herein and in the appended claims,the singular forms “a”, “an”, and “the” include plural reference unlessthe context clearly dictates otherwise. Thus, for example, reference toan “element” is a reference to one or more elements and equivalentsthereof known to those skilled in the art, and so forth.

The term “topical” refers to administration of inventive drug productat, or immediately beneath, the point of application. As used herein“topical application” refers to application onto one or more surfaces(s)including keratinous tissue, i.e., “topically applying.” Topicalapplication or “topically applying” may involve direct application tothe area of the desired substrate. The topical preparation and/orcomposition may be applied by pouring, dropping, or spraying, if aliquid; rubbing on, if an ointment, lotion, cream, gel, or the like;dusting, if a powder; spraying, if a liquid or aerosol composition; orby any other appropriate means.

As used herein, the phrase “gelling agents” refers to agents that make atopical preparation denser or more viscous in consistency. Gellingagents may be water-based or oil-based. Gelling agents may also bereferred to as “thickening agents.” “Excipients,” as used herein, areinactive ingredients that serve as carriers of the active pharmaceuticalingredient (API). Excipients may also serve as gelling or thickeningagents, or stabilizers.

A “subject” or a “mammal” includes a human or a non-human mammal.Non-human mammals include, but not limited to, livestock and pets, suchas ovine, bovine, porcine, canine, feline and murine mammals. In oneembodiment, the subject or mammal is human.

“Administering” when used in conjunction with a therapeutic means toadminister a therapeutic directly into or onto a target tissue or toadminister a therapeutic to a patient whereby the therapeutic positivelyimpacts the tissue to which it is targeted. Thus, as used herein, theterm “administering”, when used in conjunction with the drug product,can include, but is not limited to, providing the drug product and theACT peptide into or onto the target tissue. “Administering” acomposition may be accomplished by injection, topical administration, orby any method in combination with other known techniques.

Unless otherwise indicated, the term “skin” means that outer integumentor covering of the body, consisting of the dermis and the epidermis andresting upon subcutaneous tissue.

As used herein, the term “therapeutic” means an agent utilized to treat,combat, ameliorate, prevent or improve an unwanted condition or diseaseof a patient. In part, embodiments of the present invention are directedto the treatment of scarring and delayed wound healing.

As used herein, the phrase “effective amount” or “therapeuticallyeffective amount” refers to a nontoxic but sufficient amount of the ACTpolypeptides used in the practice of the invention that is effective toachieve the desired effect, i.e., to treat acute and chronic wounds. Theactivity contemplated by the present methods includes both medicaltherapeutic and/or prophylactic treatment, as appropriate, including,for example, a reduction and/or alleviation of the signs, symptoms, orcauses of a disease or disorder, or any other desired alteration of abiological system. The specific dose of a compound administeredaccording to this invention to obtain therapeutic and/or prophylacticeffects will, of course, be determined by the particular circumstancessurrounding the case, including, for example, the specific polypeptideadministered and the condition being treated. A therapeuticallyeffective amount of compound of this invention is typically an amountsuch that when it is administered in a physiologically tolerableexcipient composition, it is sufficient to achieve an effectiveintracellular concentration and local concentration in the tissue.

Generally speaking, the term “tissue” refers to any aggregation ofsimilarly specialized cells which are united in the performance of aparticular function. As used herein, “tissue”, unless otherwiseindicated, refers to tissue which includes elastin as part of itsnecessary structure and/or function.

The terms “treat,” “treated,” or “treating” as used herein refers toboth therapeutic treatment and prophylactic or preventative measures,wherein the object is to prevent, ameliorate the effects of or slow down(lessen) an undesired physiological condition, disorder or disease, orto obtain beneficial or desired clinical results. For the purposes ofthis invention, beneficial or desired clinical results include, but arenot limited to, alleviation of symptoms; diminishment of the extent ofthe condition, disorder or disease; reducing the severity of a symptomof a condition, disease or disorder; reducing the frequency of a symptomof a condition, disease or disorder; stabilization (i.e., not worsening)of the state of the condition, disorder or disease; delay in onset orslowing of the progression of the condition, disorder or disease;amelioration of the condition, disorder or disease state; and remission(whether partial or total), whether detectable or undetectable, orenhancement or improvement of the condition, disorder or disease.Treatment includes eliciting a clinically significant response withoutexcessive levels of side effects. In some embodiments, treatment mayinclude preventing or reducing scarring and/or promoting theregeneration of complex tissue structure and function following injury.

As used herein, the terms “peptide,” “polypeptide,” or “protein” areused interchangeably, and refer to a compound comprised of amino acidresidues covalently linked by peptide bonds. A protein or peptide mustcontain at least two amino acids, and no limitation is placed on themaximum number of amino acids that can comprise the sequence of aprotein or peptide. Polypeptides include any peptide or proteincomprising two or more amino acids joined to each other by peptidebonds. As used herein, the term refers to both short chains, which alsocommonly are referred to in the art as peptides, oligopeptides andoligomers, for example, and to longer chains, which generally arereferred to in the art as proteins, of which there are many types.“Polypeptides” include, for example, biologically active fragments,substantially homologous polypeptides, oligopeptides, homodimers,heterodimers, variants of polypeptides, modified polypeptides,derivatives, analogs and fusion proteins, among others. The polypeptidesinclude natural peptides, recombinant peptides, synthetic peptides or acombination thereof. A peptide that is not cyclic will have anN-terminal and a C-terminal. The N-terminal will have an amino group,which may be free (i.e., as a NH2 group) or appropriately protected (forexample, with a BOC or aFmoc group). The C-terminal will have acarboxylic group, which may be free (i.e., as a COOH group) orappropriately protected (for example, as a benzyl or a methyl ester). Acyclic peptide does not have free N- or C-terminal, since the ends arecovalently bonded through an amide bond to form the cyclic structure.Amino acids may be represented by their full names (for example,leucine), 3-letter abbreviations (for example, Leu) and 1-letterabbreviations (for example, L). The structure of amino acids and theirabbreviations can be found in the chemical literature, such as inStryer, “Biochemistry”, 3rd Ed., W. H. Freeman and Co., New York, 1988.

Excipients for use in topical gels are well-known in the art andexamples may be found in the Handbook of Pharmaceutical Excipients(Rowe, R. C. et al, APhA Publications; 5^(th) ed., 2005). Exemplaryexcipients may include waxes, various sugars and types of starch,polymers, gels, emollients, thickening agents, rheology modifiers,humectants, glycerol, organic basic compounds, cellulose derivatives,gelatin, vegetable oils, polyethylene glycols and solvents. Examples ofrheology modifiers include Carbopol, hydroxypropyl cellulose, C₂₆₋₂₈alkyl dimethicone, C₂₆₋₂₈ alkyl methicone, polyphenylsisquioxane,trimethylsiloxysilicate, crosspolymers of cyclopentasiloxane anddimethicone/vinyltrimethylsiloxysilicate, fumed silica (e.g. Cab-O-SilM5P), and mixtures thereof. Examples of emollients include glycerine,pentylene glycol, sodium pyrrolidone carboxylic acid, lanolin,saccharide isomerate, stearoxy dimethicone, stearyl dimethicone, andmixtures thereof. Emollients may be useful to prevent stratum corneumdehydration occurring due to the use of anhydrous solvents in theformulation. Examples of organic bases include 2-amino-2-methylpropanol, niacinamide, methanolamines, triethanolamines, Trisamino,AMP-95, AmP-Ultra PC 2000, triisopropanolamine, diisopropanolamine,Neutrol TE, Ethomeen, and mixtures thereof. The organic base may renderthe pH of the medicament basic or neutral.

Other exemplary excipients include water-soluble porogens. Awater-soluble porogen is an additive that may facilitate water uptakeand diffusion into the gel. Any suitable porogen may be used, but insome embodiments, the porogen may include sodium chloride, potassiumchloride, sucrose, glucose, lactose, sorbitol, xylitol, polyethyleneglycol, polyvinylpyrrollidone, polyvinyl alcohol or mixtures thereof.

Polymers may also act as excipients in topical gels. Exemplary polymersinclude hydrophilic polyurethanes, hydrophilic polyacrylates,co-polymers of carboxymethylcellulose and acrylic acid,N-vinylpyrrolidone, poly(hydroxy acids), polyanhydrides,polyorthoesters, polyamides, polycarbonates, polyalkylenes (e.g.,polyethylene and polypropylene), polyalkylene glycols (e.g.,poly(ethylene glycol)), polyalkylene oxides (e.g., polyethylene oxide),polyalkylene terephthalates (e.g., polyethylene terephthalate),polyvinyl alcohols, polyvinyl ethers, polylvinyl esters, polyvinylhalides (e.g., poly(vinyl chloride)), polyvinylpyrrolidone,polysiloxanes, poly(vinyl acetates), polystyrenes, polyurethanecopolymers, cellulose, derivatized celluloses (e.g., hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose,methylcellulose, ethylcellulose, carboxymethyl cellulose, or celluloseacetate), alginates, poly(acrylic acid), poly(acrylic acid) derivatives,acrylic acid copolymers, methacrylic acid, methacrylic acid derivatives,methacrylic acid copolymers, poly(butyric acid), poly(valeric acid),poly(lactide-co-caprolactone), copolymers thereof and blends thereof.

In some embodiments of the invention, the polymers may be superabsorbentpolymers (SAPs). A polymer is considered superabsorbent, as defined perIUPAC, as a polymer that can absorb and retain extremely large amountsof water relative to its own mass. SAPs may absorb water up to 500 timestheir own weight and may swell up to 1000-times their original volume.Particular SAPs of interest include sodium polyacrylate, thepolyurethane Tecophilic TG-2000, and polymers prepared by the use ofpolyacrylamide copolymer, ethylene maleic anhydride copolymer,cross-linked carboxy-methyl-cellulose, polyvinyl alcohol copolymers,polyvinylpyrrolindone and cross-linked polyethylene oxide.

In some embodiments of the invention, polymers that are relativelyhydrophobic may be used. Any suitable hydrophobic polymer may be used.However, exemplary polymers that are relatively hydrophobic includearomatic polyurethanes, silicone rubber, polysiloxanes,polycaprolactone, polycarbonate, polyvinylchloride, polyethylene,poly-L-lactide, poly-DL-glycolide, polyetheretherketone (PEEK),polyamide, polyimide and polyvinyl acetate. In addition, a hydrophobicgel-base and/or rheology modifier may be used.

In some embodiments of the invention, the polymers may act as thickeningagents in the medicaments. Specifically, the polymeric portion of thegel may act as a visco-elastic substance and may retain the gel at thesite of application, along with the alpha connexin polypeptidesdispersed therein.

In some other embodiments, a gel that includes a polymer may havespreadability such that it forms a thin film when applied on the skinsurface. This film may enable the application of the contained alphaconnexin polypeptides over a wide area, and may serve to maintain thealpha connexin polypeptides on the affected area of the skin.

Other excipients may include various ionic or non-ionic compounds tomaintain stability of the formulation, thereby protecting from thede-emulsification, settling, agglomeration or degradation of theformulation constituents that may reduce its therapeutic or aestheticvalue.

Examples of ionic compounds may include salts such as sodium chloride,potassium chloride; cationic, anionic or zwitterionic surfactants suchas sodium dodecyl sulfate (SDS), perfluorooctanoate (PFOA),perfluorooctanesulfonate (PFOS), ammonium lauryl sulfate (ALS), sodiumlauryl ether sulfate (SLES), alkyl benzene sulfonate, cetyltrimethylammonium bromide (CTAB), cetylpyridinium chloride (CPC),polyethoxylated tallow amine (POEA), benzalkonium chloride (BAC),benzethonium chloride, dodecyl betaine, cocamidopropyl betaine andcocoampho glycinate.

Examples of non-ionic compounds that may act as excipients includenon-ionic surfactants such as Pluronic, Tween, AMP, and Brij family ofsurfactants; and surfactants derived from biological sources, e.g,natural or semi-synthetic surfactants, such as oleic acid, sorbitantrioleate, sorbitan monooleate, lecithin, cocamide MEA, cocamide DEA andcocamidopropyl betaine. Surfactants (both ionic and non-ionic) mayreduce the interfacial surface energy and may facilitate spreading ofthe topical formulation over a wider area.

In some embodiments of the invention, solvent excipients may be used asa carrier vehicle for the alpha connexin polypeptides and otherexcipients. The polymer chains may interact with the solvent and undergoswelling to form a network that may impart visco-elastic properties tothe topical formulation. In some embodiments of the topical formulation,the solvent may evaporate upon application, leaving a residual film ofthe polymer along with the entrapped alpha connexin polypeptides.

Exemplary solvent excipients that may be useful in hydrophilicformulations may include dimethyl isosorbide, propylene glycol,glycerol, isopropanol, ethanol, benzyl alcohol, ethylene glycol,polyethylene glycol, ethoxydiglycol or mixtures thereof. Exemplarysolvent excipients that may be useful in hydrophobic formulations mayinclude capric/caprylic triglycerides, isopropyl myristate, mineral oil,isododecane, isodecyl neopentanoate, butylene glycol, pentylene glycol,hexylene glycol, methoxypolyethyleneglycol, cyclopentasiloxane,cyclotetrasiloxane, dimethicone, caprylyl methicone or mixtures thereof.

In addition to the alpha connexin polypeptides and excipients, thetopical formulation may also include at least one additional therapeuticagent such as antimicrobial agents, anti-acne agents, anti-inflammatoryagents, analgesic agents, anesthetic agents, antihistamine agents,antiseptic agents, immunosuppressants, antihemorrhagic agents,vasodilators, wound healing agents, anti-biofilm agents and mixturesthereof.

Examples of antimicrobial agents include penicillins and related drugs,carbapenems, cephalosporins and related drugs, erythromycin,aminoglycosides, bacitracin, gramicidin, mupirocin, chloramphenicol,thiamphenicol, fusidate sodium, lincomycin, clindamycin, macrolides,novobiocin, polymyxins, rifamycins, spectinomycin, tetracyclines,vanomycin, teicoplanin, streptogramins, anti-folate agents includingsulfonamides, trimethoprim and its combinations and pyrimethamine,synthetic antibacterials including nitrofurans, methenamine mandelateand methenamine hippurate, nitroimidazoles, quinolones,fluoroquinolones, isoniazid, ethambutol, pyrazinamide,para-aminosalicylic acid (PAS), cycloserine, capreomycin, ethionamide,prothionamide, thiacetazone, viomycin, eveminomycin, glycopeptide,glyclyclycline, ketolides, oxazolidinone; imipenen, amikacin,netilmicin, fosfomycin, gentamycin, ceftriaxone, Ziracin, Linezolid,Synercid, Aztreonam, and Metronidazole, Epiroprim, Sanfetrinem sodium,Biapenem, Dynemicin, Cefluprenam, Cefoselis, Sanfetrinem celexetil,Cefpirome, Mersacidin, Rifalazil, Kosan, Lenapenem, Veneprim, Sulopenem,ritipenam acoxyl, Cyclothialidine, micacocidin A, carumonam, Cefozopranand Cefetamet pivoxil.

Examples of topical anti-acne agents include adapalene, azelaic acid,benzoyl peroxide, clindamycin and clindamycin phosphate, doxycycline,erythromycin, keratolytics such as salicylic acid and retinoic acid(Retin-A”), norgestimate, organic peroxides, retinoids such asisotretinoin and tretinoin, sulfacetamide sodium, and tazarotene.Particular anti-acne agents include adapalene, azelaic acid, benzoylperoxide, clindamycin {e.g., clindamycin phosphate), doxycycline {e.g.,doxycycline monohydrate), erythromycin, isotretinoin, norgestimate,sulfacetamide sodium, tazarotene, etretinate and acetretin.

Examples of antihistamine agents include diphenhydramine hydrochloride,diphenhydramine salicylate, diphenhydramine, chlorpheniraminehydrochloride, chlorpheniramine maleate isothipendyl hydrochloride,tripelennamine hydrochloride, promethazine hydrochloride, methdilazinehydrochloride, and the like. Examples of local anesthetic agents includedibucaine hydrochloride, dibucaine, lidocaine hydrochloride, lidocaine,benzocaine, p-buthylaminobenzoic acid 2-(die-ethylamino) ethyl esterhydrochloride, procaine hydrochloride, tetracaine, tetracainehydrochloride, chloroprocaine hydrochloride, oxyprocaine hydrochloride,mepivacaine, cocaine hydrochloride, piperocaine hydrochloride, dyclonineand dyclonine hydrochloride.

Examples of antiseptic agents include alcohols, quaternary ammoniumcompounds, boric acid, chlorhexidine and chlorhexidine derivatives,iodine, phenols, terpenes, bactericides, disinfectants includingthimerosal, phenol, thymol, benzalkonium chloride, benzethoniumchloride, chlorhexidine, povidone iode, cetylpyridinium chloride,eugenol and trimethylammonium bromide.

Examples of anti-inflammatory agents include nonsteroidalantiinflammatory agents (NSAIDs); propionic acid derivatives such asibuprofen and naproxen; acetic acid derivatives such as indomethacin;enolic acid derivatives such as meloxicam, acetaminophen; methylsalicylate; monoglycol salicylate; aspirin; mefenamic acid; flufenamicacid; indomethacin; diclofenac; alclofenac; diclofenac sodium;ibuprofen; ketoprofen; naproxen; pranoprofen; fenoprofen; sulindac;fenclofenac; clidanac; flurbiprofen; fentiazac; bufexamac; piroxicam;phenylbutazone; oxyphenbutazone; clofezone; pentazocine; mepirizole;tiaramide hydrochloride; steroids such as clobetasol propionate,bethamethasone dipropionate, halbetasol proprionate, diflorasonediacetate, fluocinonide, halcinonide, amcinonide, desoximetasone,triamcinolone acetonide, mometasone furoate, fluticasone proprionate,betamethasone diproprionate, triamcinolone acetonide, fluticasonepropionate, desonide, fluocinolone acetonide, hydrocortisone vlaerate,prednicarbate, triamcinolone acetonide, fluocinolone acetonide,hydrocortisone and others known in the art, predonisolone,dexamethasone, fluocinolone acetonide, hydrocortisone acetate,predonisolone acetate, methylpredonisolone, dexamethasone acetate,betamethasone, betamethasone valerate, flumetasone, fluorometholone,beclomethasone diproprionate, fluocinonide, topical corticosteroids, andmay be one of the lower potency corticosteroids such as hydrocortisone,hydrocortisone-21-monoesters (e.g., hydrocortisone-21-acetate,hydrocortisone-21-butyrate, hydrocortisone-21-propionate,hydrocortisone-21-valerate, etc.), hydrocortisone-17,21-diesters (e.g.,hydrocortisone-17,21-diacetate, hydrocortisone-17-acetate-21-butyrate,hydrocortisone-17,21-dibutyrate, etc.), alclometasone, dexamethasone,flumethasone, prednisolone, or methylprednisolone, or may be a higherpotency corticosteroid such as clobetasol propionate, betamethasonebenzoate, betamethasone dipropionate, diflorasone diacetate,fluocinonide, mometasone furoate, triamcinolone acetonide.

Examples of analgesic agents include alfentanil, benzocaine,buprenorphine, butorphanol, butamben, capsaicin, clonidine, codeine,dibucaine, enkephalin, fentanyl, hydrocodone, hydromorphone,indomethacin, lidocaine, levorphanol, meperidine, methadone, morphine,nicomorphine, opium, oxybuprocaine, oxycodone, oxymorphone, pentazocine,pramoxine, proparacaine, propoxyphene, proxymetacaine, sufentanil,tetracaine and tramadol.

Examples of anesthetic agents include alcohols such as phenol; benzylbenzoate; calamine; chloroxylenol; dyclonine; ketamine; menthol;pramoxine; resorcinol; troclosan; procaine drugs such as benzocaine,bupivacaine, chloroprocaine; cinchocaine; cocaine; dexivacaine;diamocaine; dibucaine; etidocaine; hexylcaine; levobupivacaine;lidocaine; mepivacaine; oxethazaine; prilocaine; procaine; proparacaine;propoxycaine; pyrrocaine; risocaine; rodocaine; ropivacaine; tetracaine;and derivatives, such as pharmaceutically acceptable salts and estersincluding bupivacaine HCl, chloroprocaine HCl, diamocaine cyclamate,dibucaine HCl, dyclonine HCl, etidocaine HCl, levobupivacaine HCl,lidocaine HCl, mepivacaine HCl, pramoxine HCl, prilocaine HCl, procaineHCl, proparacaine HCl, propoxycaine HCl, ropivacaine HCl, and tetracaineHCl.

Examples of antihemorrhagic agents include thrombin, phytonadione,protamine sulfate, aminocaproic acid, tranexamic acid, carbazochrome,carbaxochrome sodium sulfanate, rutin and hesperidin.

Beside the bioactive polypeptide component, the instant invention mayalso contain other active agents such as niacinamide, phytantriol,farnesol, bisabolol and salicylic acid. It is expected that certainadditional active agents will act synergistically with the bioactivepeptide component, or will enhance the shelf-life of the formulation.

Examples of wound treatments that may be used together with the drugproduct of the present invention include fibrinolytic enzymes, such asfibrinolysin, deoxyribonuclease, streptokinase, and streptodornase,necrotomy tissue agents containing lysozyme chloride, antimicrobialagents containing gentamicin sulfate, sulfadiazine silver, bacitracin,and fradiomycin sulfate, incarnant agents containing trafermin,bucladesine sodium, tretinoin tocoferil (tocoretinate), alprostadilalfadex, solcoseryl (extract from hemolysed blood of young cattle), andalcloxa, iodine preparations containing white soft sugar, povidoneiodine, and iodine, and preparations containing bendazac, dimethylisopropylazulene (guaiazulene), and epinephrine as active ingredients.

In addition to the alpha connexin polypeptides, excipients, and othertherapeutic agents, the gels may also include other compounds thatimprove the organoleptic properties of the topical formulation.

Examples of such compounds include perfumes, dyes and colorants;chelating agents including but not limited to edetate disodium (EDTA),EGTA, CP94, citric acid; preservatives including but not limited toquaternary ammonium compounds, such as benzalkonium chloride,benzethonium chloride, cetrimide, dequalinium chloride, andcetylpyridinium chloride; mercurial agents, such as phenylmercuricnitrate, phenylmercuric acetate, and thimerosal; alcoholic agents, forexample, chlorobutanol, phenylethyl alcohol, and benzyl alcohol;antibacterial esters, for example, esters of parahydroxybenzoic acid;and other anti-microbial agents such as chlorhexidine, chlorocresol,benzoic acid and polymyxin.

Alpha connexin polypeptides that may be used in the formulation of thepresent invention are described in U.S. Pat. No. 7,786,074, which ishereby incorporated by reference. In some embodiments, the alphaconnexin polypeptide is a full length alpha connexin protein such asconnexin 37, connexin 40, connexin 43, or connexin 45. In someembodiments, the polypeptide does not comprise the full-length connexinprotein.

Peptides useful in the formulations of the present invention includealpha connexin polypeptides. The alpha connexin polypeptides useful inthe formulations of the present invention may comprise or consist of thecarboxy-terminal most 4 to 30 contiguous amino acids of an alphaconnexin protein or conservative variant thereof. Thus, the polypeptidesuseful in the formulations can comprise the c-terminal-most 4 to 30amino acids of the alpha Connexin, including the c-terminal most 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 25 22, 23,24, 25, 26, 27, 28, 29, 30 amino acids of the alpha Connexin. In someaspects, the provided polypeptide further comprises a deletion of one ormore amino acids of the c-terminal-most 4 to 30 amino acids of the alphaConnexin, including a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 aminoacids of the c-terminal-most 4 to 30 amino acids of the alpha Connexin.For example, in some aspects, the polypeptides useful in theformulations of the present invention do not comprise thec-terminal-most 1, 2, or 3 amino acids of the alpha Connexin. Forexample, the polypeptides can consist essentially of the amino acidsequence SEQ ID NO:92, or a carboxy terminal fragment thereof of atleast 4, 5, 6, 7, 8, 9, 10 amino acids in length.

In certain embodiments, the alpha connexin polypeptide of the presentinvention is linked at its amino or carboxy terminus to a cellularinternalization transporter. Separate embodiments include the alphaconnexin polypeptide without any cellular internalization transporterattached. The cellular internalization transporter linked to the alphaconnexin polypeptides may be any internalization sequence known or newlydiscovered in the art, or conservative variants thereof. Non-limitingexamples of cellular internalization transporters and sequences includeAntennapedia sequences, TAT, HIV-Tat, Penetratin, Antp-3A (Antp mutant),Buforin II, Transportan, MAP (model amphipathic peptide), K-FGF, Ku70,Prion, pVEC, Pep-1, SynB1, Pep-7, HN-1, BGSC(Bis-Guanidinium-Spermidine-Cholesterol, and BGTC(BisGuanidinium-Tren-Cholesterol).

In one embodiment of the present invention, the amino acid sequence ofthe alpha connexin polypeptide is selected from the group consisting ofSEQ ID NOs: 1, 2, 3, 4, and 5, or a conservative variant thereof. In afurther embodiment of the present invention, the alpha connexinpolypeptide comprises the amino acid sequence of SEQ ID NO: 2. Inanother embodiment of the present invention, the polypeptide comprisesan alpha connexin polypeptide and a cellular internalizationtransporter. In a further embodiment, the alpha connexin polypeptide islinked at is amino terminus to the cellular internalization transporter.In a yet further embodiment, the amino acid sequence of the alphaconnexin polypeptide linked to the cellular internalization transporteris selected from the group consisting of SEQ ID NO: 8, 9, 10, 11, and12, or a conservative variant thereof. In one embodiment, thepolypeptide consists of or comprises the amino acid sequence of SEQ IDNO: 9, namely, RQPKIWFPNRRKPWKKRPRPDDLEI.

When specific proteins are referred to herein, variants, derivatives,and fragments are contemplated. Protein variants and derivatives arewell understood to those of skill in the art and can involve amino acidsequence modifications. For example, amino acid sequence modificationstypically fall into one or more of three classes: substitutional,insertional or deletional variants. Insertions include amino and/orcarboxyl terminal fusions as well as intrasequence insertions of singleor multiple amino acid residues. Insertions ordinarily will be smallerinsertions than those of amino or carboxyl terminal fusions, forexample, on the order of one to four residues. Deletions arecharacterized by the removal of one or more amino acid residues from theprotein sequence. These variants ordinarily are prepared by sitespecific mutagenesis of nucleotides in the DNA encoding the protein,thereby producing DNA encoding the variant, and thereafter expressingthe DNA in recombinant cell culture. Techniques for making substitutionmutations at predetermined sites in DNA having a known sequence are wellknown and include, for example, M13 primer mutagenesis and PCRmutagenesis. Amino acid substitutions are typically of single residues,but can occur at a number of different locations at once; insertionsusually will be on the order of about from 1 to 10 amino acid residues.Deletions or insertions may be made in adjacent pairs, i.e., a deletionof 2 residues or insertion of 2 residues. Substitutions, deletions,insertions or any combination thereof may be combined to arrive at afinal construct. The mutations must not place the sequence out ofreading frame and preferably will not create complementary regions thatcould produce secondary mRNA structure unless such a change in secondarystructure of the mRNA is desired. Substitutional variants are those inwhich at least one residue has been removed and a different residueinserted in its place. Such substitutions generally are referred to asconservative substitutions. For example, the replacement of one aminoacid residue with another that is biologically and/or chemically similaris known to those skilled in the art as a conservative substitution. Forexample, a conservative substitution would be replacing one hydrophobicresidue for another, or one polar residue for another. The substitutionsConservatively substituted variations of each explicitly disclosedsequence are included within the polypeptides provided herein.Typically, conservative substitutions have little to no impact on thebiological activity of a resulting polypeptide. In a particular example,a conservative substitution is an amino acid substitution in a peptidethat does not substantially affect the biological function of thepeptide. A peptide can include one or more amino acid substitutions, forexample 2-10 conservative substitutions, 2-5 conservative substitutions,4-9 conservative substitutions, such as 2, 5 or 10 conservativesubstitutions. A polypeptide can be produced to contain one or moreconservative substitutions by manipulating the nucleotide sequence thatencodes that polypeptide using, for example, standard procedures such assite-directed mutagenesis or PCR. Alternatively, a polypeptide can beproduced to contain one or more conservative substitutions by usingstandard peptide synthesis methods. An alanine scan can be used toidentify which amino acid residues in a protein can tolerate an aminoacid substitution. In one example, the biological activity of theprotein is not decreased by more than 25%, for example not more than20%, for example not more than 10%, when an alanine, or otherconservative amino acid (such as those listed below), is substituted forone or more native amino acids. Further information about conservativesubstitutions can be found in, among other locations, in Ben-Bassat etal., (J. Bacterial. 169:751-7, 1987), O'Regan et al., (Gene 77:237-51,1989), Sahin-Toth et al., (Protein Sci. 3:240-7, 1994), Hochuli et al.,(Bio/Technology 6:1321-5, 1988) and in standard textbooks of geneticsand molecular biology.

As used herein, “analytical methods” includes the following techniquesthat are useful for determining the identity and quantity of the ACTpeptide in a particular formulation: NMR, HPLC, Amino Acid Compositionand Molecular Weight Determinations, and Specific Optical Rotation.

The present invention includes a topical formulation comprising at leastone alpha connexin polypeptide and hydroxyethylcellulose gel, whereinthe hydroxyethylcellulose gel stabilizes the alpha connexin polypeptide.In certain embodiments, the hydroxyethylcellulose gel stabilizes thealpha connexin polypeptide so that after 3 months of storage at 5° C. atleast 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the alpha connexinpolypeptide is detectable by analytical methods. In some embodiments,the alpha connexin polypeptide is present in the formulation at aconcentration of about 0.0025% (w/w), of about 0.005% (w/w), of about0.0075% (w/w), of about 0.010% (w/w), of about 0.015% (w/w), of about0.020% (w/w), of about 0.025% (w/w), of about 0.030% (w/w), of about0.035% (w/w), of about 0.040% (w/w), of about 0.045% (w/w), of about0.050% (w/w), of about 0.055% (w/w), of about 0.060% (w/w), of about0.065% (w/w), of about 0.070% (w/w), of about 0.075% (w/w), of about0.080% (w/w), of about 0.085% (w/w), of about 0.090% (w/w), of about0.095% (w/w), of about 0.100% (w/w), of about 0.150% (w/w), of about0.200% (w/w), of about 0.250% (w/w), of about 0.500% (w/w), of about0.750% (w/w), of about 1.00% (w/w), of about 1.50% (w/w), of about 2.00%(w/w), of about 2.50% (w/w), or of about 5.00 (w/w). In one embodiment,the alpha connexin polypeptide is present in the formulation at aconcentration of between about 0.005% (w/w) and about 1.00% (w/w).

In other embodiments, the drug product of the invention is a clearcolorless gel which contains 0.0072% (w/w) (20 μM) of the ACT peptide,0.018% (w/w) (50 μM) of the ACT peptide, 0.036% (w/w) (100 μM) of theACT peptide, or 0.072% (w/w) (200 μM) of the ACT peptide. The ACTpeptide may be dissolved in a semisolid dosage form that contains >0%water, >10% water, >20% water, >30% water, >40% water, >50% water, >60%water, >70% water, >80% water, or >90% water and 0.25% gelling agent(polymer), 0.55% gelling agent (polymer), 0.75% gelling agent (polymer),1.00% gelling agent (polymer), 1.25% gelling agent (polymer), 1.50%gelling agent (polymer), 1.75% gelling agent (polymer), 2.00% gellingagent (polymer), 2.25% gelling agent (polymer), or 2.50% gelling agent(polymer). The ACT peptide may be well preserved and adequately bufferedto pH 6. In one embodiment of the topical formulation, the qualitativeand quantitative composition is that listed in Table 1.

TABLE 1 Drug Product Gel Qualitative & Quantitative CompositionConcentration Ingredients Grade Function (% w/w) Peptide 328967 — Active0.0072; 0.018 (ACT peptide) 0.036; 0.072 Methylparaben NF Preservative0.17 Propylparaben NF Preservative 0.02 Glycerin USP Solvent 5.0 SodiumPhosphate USP Buffer Agent 0.263 Monobasic Sodium Phosphate Dibasic USPBuffer Agent 0.044 Propylene Glycol USP Solvent 3.0 Edetate Disodium(EDTA) USP Chelating Agent 0.05 D-Mannitol USP Stabilizer 0.05Hydroxyethylcellulose, NF Gelling Agent 1.25%  250HHX Purified Water,qsad USP Solvent 100%

The ACT1 peptide sequence is listed in Table 2 below in which Ahx refersto L-2-aminohexanoic acid, also known as 6-aminohexanoic acid:

TABLE 2 Peptide 328967 (Antp/ACT1) sequence.Biotin-Ahx-Arg-Gln-Pro-Lys-lle-Trp-Phe-Pro-Asn-Arg-Arg-Lys-Pro-Trp-Lys-Lys-Arg-Pro-Arg-Pro-Asp- Asp-Leu-Glu-lle-OH

The general properties of the Peptide 328967 are listed in Table 3.

TABLE 3 General Physical Properties of Peptide 328967 PhysicalAppearance White to off-white powder Molecular Weight 3597.33 ± 2.0 amuCounter-Ion AcOH Solubility Soluble in water at room temperature

In one aspect of the invention, the excipients used in the drug producttopical preparation are selected from the group consisting of or are oneor more of the following:

Methylparaben

Propylparaben

Glycerin

Sodium Phosphate Monobasic

Sodium Phosphate Dibasic

Propylene Glycol

Edetate Disodium (EDTA)

D-Mannitol

Hydroxyethylcellulose, 250 HHX

Purified Water

In one embodiment, the drug product topical preparation comprises apeptide, D-mannitol, hydroxyethylcellulose, and purified water. Saidpreparation may further comprise one or more of methylparaben,propylparaben, glycerin, sodium phosphate monobasic, sodium phosphatedibasic, propylene glycol, and edetate disodium (EDTA). In a furtherembodiment, the hydroxyethylcellulose is 250 HHX. In a furtherembodiment, the peptide is an alpha connexin polypeptide.

In such embodiments, the drug product topical preparation comprises apeptide at a concentration between about 0.001% (w/w) and about 0.5%(w/w) (for example, at about 0.0072%, 0.018%, 0.036%, or 0.072% (w/w));methylparaben at a concentration between about 0.10% (w/w) and about0.25% (w/w) (for example, about 0.17% (w/w)); propylparaben at aconcentration between about 0.01% (w/w) and about 0.03% (w/w) (forexample, about 0.02% (w/w)); glycerin at a concentration between about1% (w/w) and about 10% w/w) (for example, about 5% (w/w)); sodiumphosphate monobasic at a concentration between about 0.1% (w/w) andabout 0.5% (w/w) (for example, about 0.263% (w/w)); sodium phosphatedibasic at a concentration between about 0.02% and about 0.06% (forexample, about 0.044% (w/w)); propylene glycol at a concentrationbetween about 1% (w/w) and about 5% (w/w) (for example, about 3% (w/w));EDTA at a concentration between about 0.01% and about 0.1% (for example,about 0.05% (w/w)); D-mannitol at a concentration between about 0.01%(w/w) and about 0.1% (w/w) (for example, about 0.05% (w/w));hydroxyethylcellulose at a concentration between about 0.5% and about2.5% (for example, about 1.25% (w/w)), and purified water at aconcentration of about 0.1% to about 10% (for example, about 1%). In afurther embodiment, the peptide is an alpha connexin polypeptide. In ayet further embodiment, the peptide comprises an amino acid sequenceselected from the group consisting of SEQ ID NO: 1, 2, 3, 4, 5, 8, 9,10, 11, and 12. In one embodiment, the peptide comprises an amino acidsequence according to SEQ ID NO: 9.

Derived through in vitro and in vivo studies, these stabilizers andexcipients are incorporated into the drug product since they arenon-irritating, non-staining, and non-immunogenic. Stability studiesshowed that the ACT1 peptide is more stable in the gelling agentHydroxyethylcellulose, 250 HHX (1.25%) compared to Pluronic gels. TheACT peptide within the drug product containing 1.25%hydroxyethylcellulose only dropped to 98% of the label claim (i.e.,initial concentration) when stored at 5° C. for three months and to 84%of the label claim when stored at 25° C. for the same duration. In oneaspect of the invention, edetate disodium (EDTA) and Mannitol areincorporated within the drug product to provide stability to the ACT1peptide. In some embodiments of the invention, the Mannitol is presentin the formulation at 0.01% (w/w) to 1.6% (w/w), 0.01% (w/w) to 1.5%(w/w), 0.01% (w/w) to 1.4% (w/w), 0.01% (w/w) to 1.3% (w/w), 0.01% (w/w)to 1.2% (w/w), 0.01% (w/w) to 1.1% (w/w), 0.01% (w/w) to 1.0% (w/w),0.01% (w/w) to 0.9% (w/w), 0.01% (w/w) to 0.8% (w/w), 0.01% (w/w) to0.7% (w/w), 0.01% (w/w) to 0.6% (w/w), 0.01% (w/w) to 0.5% (w/w), 0.01%(w/w) to 0.4% (w/w), 0.01% (w/w) to 0.3% (w/w), 0.01% (w/w) to 0.2%(w/w), 0.01% (w/w) to 0.1% (w/w), or 0.01% (w/w) to 0.0.05% (w/w). Inone embodiment, the Mannitol is present in the formulation at about0.05% (w/w).

In another aspect of the present invention, a buffering agent isincluded in the topical formulation to maintain the pH in a certainrange. Suitable buffering agents can include, but are not limited to,acetate buffers, citrate buffers, phosphate buffers, lactic acidbuffers, malic acid buffers, succinic acid buffers, borate buffers,sodium hydroxide, potassium hydroxide, and ammonium hydroxide. Phosphatesalts such as monosodium phosphate (NaH₂PO₄; also known as monobasicsodium phosphate), disodium hydrogen phosphate (Na₂HPO₄; also known asdibasic sodium phosphate), monopotassium phosphate (KH₂PO₄), dipotassiumphosphate (K₂HPO₄), and mixtures thereof can also be used. In oneembodiment, phosphate buffer provides superior stability compared tocitrate buffer. A buffer capacity at 25 mM was found to be adequate forthe drug product. Buffer is needed to control pH of the gel system andto maintain stability of the peptide drug. The pH range of the topicalformulation may be pH 2 to pH 12, pH 4 to pH 10, or pH 6 to pH 8. In oneembodiment, the proper pH range is in the range of 5 to 7. In someembodiments, the pH of the topical formulation of the present inventionis 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, or 8.0. In yet anotheraspect, propylene glycol in the amount of 3% provides bettersolubilization of parabens in the aqueous system.

In one embodiment, the formulation with an ACT peptide incorporated intohydroxyethylcellulose enables large scale manufacturing of a productwith the characteristics that make it practical for clinical treatmentsas well as meeting desired storage and stability requirements. While aformulation for ACT 1 using Pluronic gel may require a relatively longincorporation time of approximately 2.5 hours and only yield 50 grambatches, a formulation with hydroxyethylcellulose provides allows forsignificantly faster incorporation of the ACT peptide into the gel andyields much larger batches. For example, when using Pluronic F 127 gelin the topical formulation it takes over an hour to incorporate thepolymer, and the formulation needs to be placed in a water bath to helpwith incorporation. In contrast, hydroxyethlycellulose (e.g., HEC 250HHX) is easily incorporated at room temperature and hydrates within 30minutes. Thus, the use of hydroxyethylcellulose may facilitate largescale manufacturing. The manufacturing process with Pluronic gel mayrequire a cold bath to bring its viscosity into a desired range andrequire more energy than the manufacturing process withhydroxyethylcellulose. In addition, the final formulation in Pluronicgel may be very thin at a storage condition of 5 degrees C.

It has been discovered that hydroxyethylcellulose (HEC) is a suitablegelling agent and acceptable carrier of the drug product of the presentinvention. In one embodiment, the gelling agent is Hydroxyethylcellulose(HEC), 250 HHX. In one embodiment, the percent (w/w) of HEC is in therange of 1-5%. In a further embodiment, the percent (w/w) of HEC is1.25%. In the manufacture of HEC, a purified cellulose is reacted withsodium hydroxide to produce a swollen alkali cellulose. Thealkali-treated cellulose is more chemically reactive than cellulose. Byreacting the alkali cellulose with ethylene oxide, a series ofhydroxyethylcellulose ethers is produced. In this reaction, the hydrogenatoms in the hydroxyl groups of cellulose are replaced by hydroxyethylgroups, which confer water solubility to the gel. It is contemplated inthis invention that a single HEC ether may be used, or a mixture of HECethers of difference molecular weight and structure may be used.Suitable grades of HEC for pharmaceutical purposes are well known andfull described in the pharmaceutical literature. Suitable commerciallyavailable brands of HEC include but are not limited to Fuji HEC-HP; FujiHEC-AG 15; NATRO-SOL 250HR; NATROSOL 250MH; NATROSOL 250G; CELLOSIZE QP30000; TYLOSE H SERIES; NATROSOL 180L; NATROSOL 300H; TYLOSE P-X;NATROSOL 250M; CELLOSIZE WP 4400; CELLOSIZE UT 40; NATROSOL 250H4R;Tylose H 20P; NATROSOL LR; TYLOSE MHB; NATROSOL 250HHP; HERCULES N 100;CELLOSIZE WP 300; TYLOSE P-Z SERIES; NATROSOL 250H; TYLOSE PS-X;Cellobond HEC 400; CELLOSIZE QP; CELLOSIZE QP 1500; NATRO-SOL 250;HYDROXYETHYL CELLULOSE ETHER; HESPAN; TYLOSE MHB-Y; NATROSOL 240JR;HYDROXYETHYL STARCH; CELLOSIZE WP; CELLOSIZE WP 300H; 2-HYDROXYETHYLCELLULOSE ETHER; BL 15; CELLOSIZE QP 4400; CELLOSIZE QP3; TYLOSE MB;CELLULOSE HYDROXY-ETHYLATE; CELLOSIZE WPO 9H17; CELLOSIZE 4400H16;CELLULOSE HYDROXYETHYL ETHER; Hydroxyethyl Cellulose; Hydroxyl EthylCellulose (HEC); Hydroxyethyl Cellulose 100H (celocell 100h); TYLOSEMH-XP; NATROSOL 250HX; Natrosol; Daicel EP 500; HEC-Unicel; HEC(Hydroxyethyl cellulose); Cellosize; HEC-Al 5000; Fuji HEC-AL 15;HEC-Unicel QP 09L; Cellulose, ethers, 2-hydroxyethyl ether; Unicel QP52000H; HEC-QP 4400; SP 250 (cellulose); Hetastarch; Cellulose, ethers,2-hydroxyethyl ether; Glutofix 600; FL 52; Fuji HEC-AX 15F; Tylose H300P; HEC-Unicel QP 300H; Tylose H 300; Daicel SP 550; Daicel SE 600;Unicel QP 15000; HEC-QP 100MH; HEC-QP 9H; OETs; Daicel EP 850; H. E.Cellulose; Cellobond 25T; Unicel QP 100MH; Tylose H 4000; SE 850K;Tylomer H 20; Daicel SE 850K; Tylose H 30000YP; Unicel QP 4400; SP 407;Tylose H 100000; Daicel SP 200; Culminal HEC 5000PR; Tylopur H 300;Daicel SP 750; Sanhec; BL 15 (cellulose derivative); Unicel QP 300H;Tylomer H 200; J 164; Tylose H 10; Tylose H 20; AH 15; Daicel SP 600;Daicel SE 900; HEC-Unicel QP 4400H; AX 15; Daicel SP 800; Fuji HEC-AW15F; HEC-SE 850; HEC-A 5-25CF; Metolose 90SEW; AW 15 (polysaccharide);Cellobond HEC 5000; HEC-QP 100M; Cellobond HEC 15A; Tylose H 15000YP2;Walocel HT 6.000PFV; 2-Hydroxyethyl cellulose (Natrosol Type 250HRCS);Fuji HEC-BL 20; Fuji HEC-SY 25F; Telhec; HEC-SP 200; HEC-AH 15;HEC-Unicel QP 30000H; see; HEC 10A; Daicel SP 400; Admiral 3089FS; FujiHEC-A 5000F; HEC-SP 400; Hydroxyethyl Methyl Cellulose (HEMC);HYDROXYETHYL CELLULOSE (HEC); Hydroxyethyl Starch (CAS No: 9004-62-0);Hydroxy Ethyl Cellulose; “Natrosol” [Aqualon]; HEC; 2-HYDROXYETHYLCELLULOSE; NATROSOL 150L; TYLOSE MHB-YP; HYDROXYETHYL ETHER CELLULOSE;NATROSOL 250L; CELLOSIZE WP 400H; TYLOSE P; CELLULOSE, 2-HYDROXYETHYLETHER; TYLOSE MH-K; NATROSOL 250HHR.

In another embodiment, the drug product of the present invention ispackaged in 20 mL vial (USP Type I, Borosilicate clear scintillationglass with poly-seal cone urea screw cap). A mixture of methylparaben at0.17% (w/w) and propylparaben 0.02% (w/w) is used as a preservative.

In some embodiments, the present invention includes a method of woundtreatment comprising administering to a subject in need thereof atopical formulation comprising at least one alpha connexin polypeptideand hydroxyethylcellulose gel, wherein the hydroxyethylcellulose gelstabilizes the alpha connexin polypeptide. The wound treated may be anacute surgical wound or a chronic, non-infected, full-thickness lowerextremity ulcer.

In a certain embodiment, the drug product of the present invention maybe used to mitigate excessive scar formation associated with acutesurgical wounds. In this embodiment, the drug product of the presentinvention may be applied at the time of surgical incision closure, 1hour after surgical incision closure, 2 hours after surgical incisionclosure, 3 hours after surgical incision closure, 4 hours after surgicalincision closure, 5 hours after surgical incision closure, 6 hours aftersurgical incision closure, 7 hours after surgical incision closure, 8hours after surgical incision closure, 9 hours after surgical incisionclosure, 10 hours after surgical incision closure, 11 hours aftersurgical incision closure, 12 hours after surgical incision closure, 13hours after surgical incision closure, 14 hours after surgical incisionclosure, 15 hours after surgical incision closure, 16 hours aftersurgical incision closure, 17 hours after surgical incision closure, 18hours after surgical incision closure, 19 hours after surgical incisionclosure, 20 hours after surgical incision closure, 21 hours aftersurgical incision closure, 22 hours after surgical incision closure, 23hours after surgical incision closure, 24 hours after surgical incisionclosure, 48 hours after surgical incision closure, 72 hours aftersurgical incision closure, or thereafter.

In another embodiment, the drug product of the present invention may beused to treat chronic ulcers. For example, ulcers may include diabeticfoot ulcers, venous leg ulcers, and pressure ulcers. These ulcers may bechronic, non-infected, full-thickness lower extremity ulcers. In oneembodiment, the drug product of the present invention may be applied toa chronic ulcer in a daily regimen, a regimen of every other day, aregimen of once a week, or in various other regimens until healing ofthe chronic ulcer is apparent. In another embodiment, the drug productof the present invention may be applied to a chronic ulcer in a regimenat day 0, 3, 7, 14, 21, and 28. In another embodiment, the drug productof the present invention may be applied to a chronic ulcer in a regimenat day 0, day 3, week 1, week 2, week 3, week 4, week 5, week 6, week 7,week 8, week 9, week 10, week 11, and week 12. In another aspect of thepresent invention, the drug product is manufactured with the followingsteps:

Step 1: In a suitable size of beaker, add propylene glycol, glycerin,methylparaben and propylparaben. Mix with a propeller until the parabensare completely dissolved.

Step 2: In a manufacturing vessel, add purified water (part I), EDTA,monobasic sodium phosphate, dibasic sodium phosphate and D-mannitol. Mixwith a propeller until a clear solution is obtained.

Step 3: Add the solution from step 1 to the manufacturing vessel. Rinsethe beaker with purified water (part II, divided into approximately 3equal portions) and add the rinse back to the vessel. Continue withpropeller mixing until the solution is visually homogeneous.

Step 4: With homogenization mixing, add hydroxyethyl cellulose into themanufacturing vessel from Step 3. Mix until the polymer is fullydispersed.

Step 5: In a separate beaker, add purified water (part III) and an alphaconnexin polypeptide (e.g., Peptide 328967 ACT1 peptide). Mix with astir bar or propeller mixer until the peptide is completely dissolvedand a gel is formed.

Step 6: With continuous propeller mixing, add the drug solution fromstep 5 to the manufacturing vessel. Rinse the beaker with purified water(part IV, divided into approximately 3 equal portions) and add the rinseback to the vessel. Mix until the gel is homogeneous.

The manufacturing process flow chart is provided in FIG. 1 ManufacturingProcess Flow Chart.

Sequences

(ACT 2) SEQ ID NO: 1 PSSRASSRASSRPRPDDLEI (ACT 1) SEQ ID NO: 2 RPRPDDLEI(ACT 3) SEQ ID NO: 3 RPRPDDLEV (ACT 4) SEQ ID NO: 4 RPRPDDVPV (ACT 5)SEQ ID NO: 5 KARSDDLSV SEQ ID NO: 6 aga cct cgg cct gat gac ctg gag att(Antp) SEQ ID NO: 7 RQPKIWFPNRRKPWKK (Antp/ACT 2) SEQ ID NO: 8RQPKIWFPNRRKPWKKPSSRASSRASSRPRPDDLEI (Antp/ACT 1) SEQ ID NO: 9RQPKIWFPNRRKPWKKRPRPDDLEI (Antp/ACT 3) SEQ ID NO: 10RQPKIWFPNRRKPWKKRPRPDDLEV (Antp/ACT 4) SEQ ID NO: 11RQPKIWFPNRRKPWKKRPRPDDVPV (Antp/ACT 5) SEQ ID NO: 12RQPKIWFPNRRKPWKKKARSDDLSV (encodes polypeptide of SEQ ID NO 9)SEQ ID NO: 13  cgg cag ccc aag atc tgg ttc ccc aac cgg cggaag ccc tgg aag aag cgg ccc ggc ccg acg acc tgg aga tc (HIV-Tat)SEQ ID NO: 14 GRKKRRQRPPQ (Penetratin) SEQ ID NO: 15 RQIKIWFQNRRMKWKK(Antp-3A) SEQ ID NO: 16 RQIAIWFQNRRMKWAA (Tat) SEQ ID NO: 17 RKKRRQRRR(Buforin II) SEQ ID NO: 18 TRSSRAGLQFPVGRVHRLLRK (Transportan)SEQ ID NO: 19 GWTLNSAGYLLGKINKALAALAKKIL (model amphipathic peptide)SEQ ID NO: 20 KLALKLALKALKAALKLA (K-FGF) SEQ ID NO: 21 AAVALLPAVLLALLAP(Ku70) SEQ ID NO: 22 VPMLK-PMLKE (Prion) SEQ ID NO: 23MANLGYWLLALFVTMWTDVGLCKKRPKP (pVEC) SEQ ID NO: 24 LLIILRRRIRKQAHAHSK(Pep-1) SEQ ID NO: 25 KETWWETWWTEWSQPKKKRKV (SynB1) SEQ ID NO: 26RGGRLSYSRRRFSTSTGR (Pep-7) SEQ ID NO: 27 SDLWEMMMVSLACQY (HN-1)SEQ ID NO: 28 TSPLNIHNGQKL (Chick alpha C × 43 ACT) SEQ ID NO: 29PSRASSRASSRPRPDDLEI (Human alpha C × 45) SEQ ID NO: 30GSNKSTASSKSPDPKNSVWI (Chick alpha C × 45) SEQ ID NO: 31GSNKSSASSKSGDGKNSVWI (Human alpha C × 46) SEQ ID: 32GRASKASRASSGRARPEDLAI (Human alpha C × 46.6) SEQ ID: 33 GSASSRDGKTVWI(Chimp alpha C × 36) SEQ ID NO: 34 PRVSVPNFGRTQSSDSAYV (Chick alpha C ×36) SEQ ID NO: 35 PRMSMPNFGRTQSSDSAYV (Human alpha C × 47) SEQ ID NO: 36PRAGSEKGSASSRDGKTTVWI (Human alpha C × 40) SEQ ID NO: 37GYHSDKRRLSKASSKARSDDLSV (Human alpha C × 50) SEQ ID NO: 38PLSRLSKASSRARSDDLTV (Human alpha C × 59) SEQ ID NO: 39PNHVVSLTNNLIGRRVPTDLQI (Rat alpha C × 33) SEQ ID NO: 40PSCVSSSAVLTTICSSDQVVPVGLSSFYM (Sheep alpha C × 44) SEQ ID NO: 41GRSSKASKSSGGRARAADLAI (Human beta C × 26) SEQ ID NO: 42LCYLLIRYCSGKSKKPV (Human alpha C × 37) SEQ ID: 43 GQKPPSRPSSSASKKQ*YVSEQ ID 44: (conservative C × 43 variant) SSRASSRASSRPRPDDLEVSEQ ID 45: (conservative C × 43 variant) RPKPDDLEISEQ ID 46: (conservative C × 43 variant) SSRASSRASSRPKPDDLEI,SEQ ID 47: (conservative C × 43 variant) RPKPDDLDISEQ ID 48: (conservative C × 43 variant) SSRASSRASSRPRPDDLDISEQ ID 49: (conservative C × 43 variant) SSRASTRASSRPRPDDLEISEQ ID 50: (conservative C × 43 variant) RPRPEDLEISEQ ID 51: (conservative C × 43 variant) SSRASSRASSRPRPEDLEI,SEQ ID 52: (conservative C × 45 variant) GDGKNSVWVSEQ ID 53: (conservative C × 45 variant) SKAGSNKSTASSKSGDGKNSVWVSEQ ID 54: (conservative C × 37 variant) GQKPPSRPSSSASKKLYV(non-active control peptide) SEQ ID NO: 55 RQPKIWFPNRRKPWKIELDDPRPR(HIV-Tat/ACT 1) SEQ ID NO: 56 GRKKRRQRPPQRPRPDDLEI (Penetratin/ACT 1)SEQ ID NO: 57 RQIKIWFQNRRMKWKKRPRPDDLEI (Antp-3A/ACT 1) SEQ ID NO: 58RQIAIWFQNRRMKWAARPRPDDLEI (Tat/ACT 1) SEQ ID NO: 59 RKKRRQRRRRPRPDDLEI(Buforin II/ACT 1) SEQ ID NO: 60 TRSSRAGLQFPVGRVHRLLRKRPRPDDLEI(Transportan/ACT 1) SEQ ID NO: 61 GWTLNSAGYLLGKINKALAALAKKILRPRPDDLEI(MAP/ACT 1) SEQ ID NO: 62 KLALKLALKALKAALKLARPRPDDLEI (K-FGF/ACT 1)SEQ ID NO: 63 AAVALLPAVLLALLAPRPRPDDLEI (Ku70/ACT 1) SEQ ID NO: 64VPMLKPMLKERPRPDDLEI (Prion/ACT 1) SEQ ID NO: 65MANLGYWLLALFVTMWTDVGLCKKRPKPRPRPDDLEI (pVEC/ACT 1) SEQ ID NO: 66LLIILRRRIRKQAHAHSKRPRPDDLEI (Pep-1/ACT 1) SEQ ID NO: 67KETWWETWWTEWSQPKKKRKVRPRPDDLEI (SynB1/ACT 1) SEQ ID NO: 68RGGRLSYSRRRFSTSTGRRPRPDDLEI (Pep-7/ACT 1) SEQ ID NO: 69SDLWEMMMVSLACQYRPRPDDLEI (HN-1/ACT 1) SEQ ID NO: 70TSPLNIHNGQKLRPRPDDLEI (20 to 120 residues flanking amino acid 363 ofhuman C × 43) SEQ ID NO: 71KGKSDPYHATSGALSPAKDCGSQKYAYFNGCSSPTAPLSPMSPPGYKLVTGDRNNSSCRNYNKQASEQNWANYSAEQNRMGQAGSTISNSHAQPFDFPDDNQNSKKLAAGHELQPLAIVDQR(20 to 120 residues flanking amino acid 362 of  chick C × 43)SEQ ID NO: 72 KTDPYSHSGTMSPSKDCGSPKYAYYNGCSSPTAPLSPMSPPGYKLVTGDRNNSSCRNYNKQASEQNWANYSAEQNRMGQAGSTISNSHAQPFDFADEHQNTKKLASGHELQPLTIVDQRP(20 to 120 residues flanking amino acid 377 of   human C × 45)SEQ ID NO: 73 LGFGTIRDSLNSKRRELEDPGAYNYPFTWNTPSAPPGYNIAVKPDQIQYTELSNAKIAYKQNKANTAQEQQYGSHEENLPADLEALQREIRMAQERLDLAVQAYSHQNNPHGPREKKAKV (20 to 120 residues flanking amino acid 375 of chick C × 45) SEQ ID NO: 74GFGTIRDTLNNKRKELEDSGTYNYPFTWNTPSAPPGYNIAVKPDQMQYTELSNAKMAYKQNKANIAQEQQYGSNEENIPADLENLQREIKVAQERLDMAIQAYNNQNNPGSSSREKKSKA.(20 to 120 residues flanking amino acid 313 of   human C × 37)SEQ ID NO: 75 PYLVDCFVSRPTEKTIFIIFMLVVGLISLVLNLLELVHLLCRCLSRGMRARQGQDAPPTQGTSSDPYTDQVFFYLPVGQGPSSPPCPTYNGLSSSEQNWANLTTEERLASSRPPLFLDPP (20 to 120 residues flanking amino acid 258 of rat C × 33) SEQ ID NO: 76CGSKEHGNRKMRGRLLLTYMASIFFKSVFEVAFLLIQWYLYGFTLSAVYICEQSPCPHRVDCFLSRPTEKTIFILFMLVVSMVSFVLNVIELFYVLFKAIKNHLGNEKEEVYCNPVELQK. (enhanced green fluorescent protein)SEQ ID NO: 77 MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK (ACT 2) SEQ ID NO: 78CCCTCCTCCCGGGCCTCCTCCCGGGCCTCCTCCCGGCCCCGGCCCGAC GACCTGGAGATC (ACT 1)SEQ ID NO: 79 CGGCCCCGGCCCGACGACCTGGAGATC (ACT 3) SEQ ID NO: 80CGGCCCCGGCCCGACGACCTGGAGGTG (ACT 4) SEQ ID NO: 81CGGCCCCGGCCCGACGACGTGCCCGTG (ACT 5) SEQ ID NO: 82AAGGCCCGGTCCGACGACCTGTCCGTG (Antp) SEQ ID NO: 83CGGCAGCCCAAGATCTGGTTCCCCAACCGGCGGAAGCCCTGGAAG  AAG (Antp/ACT 2)SEQ ID NO: 84 CGGCAGCCCAAGATCTGGTTCCCCAACCGGCGGAAGCCCTGGAAGAAGCCCTCCTCCCGGGCCTCCTCCCGGGCCTCCTCCCGGCCCCGGCCCGAC GACCTGGAGATC(Antp/ACT 1) SEQ ID NO: 85CGGCAGCCCAAGATCTGGTTCCCCAACCGGCGGAAGCCCTGGAAGAAGCGGCCCCGGCCCGACGACCTGGAGATC (Antp/ACT 3) SEQ ID NO: 86CGGCAGCCCAAGATCTGGTTCCCCAACCGGCGGAAGCCCTGGAAGAAGCGGCCCCGGCCCGACGACCTGGAGGTG (Antp/ACT 4) SEQ ID NO: 87CGGCAGCCCAAGATCTGGTTCCCCAACCGGCGGAAGCCCTGGAAGAAGCGGCCCCGGCCCGACGACGTGCCCGTG (Antp/ACT 5) SEQ ID NO: 88CGGCAGCCCAAGATCTGGTTCCCCAACCGGCGGAAGCCCTGGAAGAAGAAGGCCCGGTCCGACGACCTGTCCGTG (Zebrafish alpha C × 43) SEQ ID NO: 89PCSRASSRMSSRARPDDLDV (Chick alpha C × 36) SEQ ID NO: 90PRVSVPNFGRTQSSDSAYV (Zebrafish alpha C × 36) SEQ ID NO: 91PRMSMPNFGRTQSSDSAYV (C × 43 isoleucine deletion) SEQ ID NO: 92RQPKIWFPNRRKPWKKRASSRASSRPRPDDLE

EXAMPLES Example 1

The objective was to develop a stable, elegant and pourable gel in whichthe Active Pharmaceutical Ingredient (API) is in solution for woundhealing purposes.

The major challenges encountered during formulation development were theselection of suitable stabilizers for the API (ACT1), and otherexcipients that are non-irritating for wound healing application.Studies were conducted to evaluate prototype formulations.

R&D Study Per Protocol #1062

The first study was conducted per protocol #1062. During this study, 6prototype formulations were evaluated as outlined in Table 4. Thepurposes of the study were to select a proper pH range for the productand also to select a proper gelling agent. The API concentration usedfor this study was 0.072% which was the initial targeted productconcentration. Phosphate buffers at pH 5, 6, and 7 and two gellingagents, Pluronic and Hydroxyethylcellulose (HEC) were evaluated. Thequantity of API required for the formulations was calculated as follows:

Batch size for each formulation=12×5 g=60 g or 70 g

API quantity for 6 formulations=6×70×0.072%/82.4%=0.37 g

API purity value=82.4%

Five formulations, formulae 2414-10 to -14, were gelled with Pluronic127, which was assumed to be the preferred gelling agent. Only oneformulation, 2414-15, was gelled with HEC as a back-up. The availablestability data indicated the following conclusions.

Buffer was needed to control the pH of the gel system and to maintainstability of the peptide drug. Proper pH range seemed to be between 5and 7. Phosphate buffer was adequate.

Surprisingly, the API, ACT1, was more stable in HEC gel than Pluronicgels. With Pluronic gels, the API dropped to an average of 85% of thelabel claim (LC) from initial when gels were stored at 5° C. for onemonth; and to 79% when stored at 25° C. for one month. With HEC gel, theAPI dropped to 98% of label claim from initial when stored at 5° C. for3 months and 84% when stored at 25° C. for three months.

HEC appeared to be a better choice of gelling agent than Pluronic whencomparing formula 2414-14 to 2414-15. Both formulations were exactlysame except that formula 2414-14 was gelled with Pluronic and formula2414-15 was gelled with HEC. The API in formula 2414-14 dropped to 68%of the label claim from the initial concentration when stored at 5° C.for one month while the API in formula 2414-15 only dropped to 98% ofthe label claim from the initial concentration when stored at 5° C. for3 months.

EDTA and Mannitol appear to provide some stability to the peptide drug.

The formulations did not appear to be stable at 25° C. The final productmight require refrigeration, which is typical for products containingpeptides and proteins.

Formula 2414-15 with HEC as the gelling agent is the most stableformulation compared to the rest of the formulations tested. It wasselected for further development. Stability data of formulations 2414-14and 2414-15 are summarized in Tables 5 and 6, respectively.

TABLE 4 Wound Healing Gel Formulation Compositions for Stability Testingper Study #1062 Components Formula #: Formula #: Formula #: Formula #:Formula #: Formula #: (% W/W) 2414-10 2414-11 2414-12 2414-13 2414-142414-15 API 0.072 0.072 0.072 0.072 0.072 0.072 Propylene glycol 1.0 1.01.0 1.0 1.0 1.0 Glycerin 5.0 5.0 5.0 5.0 5.0 5.0 Monobasic Sodium —0.300 0.263 0.120 0.263 0.263 Phosphate Dibasic Sodium — 0.0036 0.0440.217 0.044 0.044 Phosphate Methylparaben 0.17 0.17 0.17 0.17 0.17 0.17Propylparaben 0.02 0.02 0.02 0.02 0.02 0.02 Pluronic 127 20.0 20.0 20.020.0 20.0 — (Poloxmer 407) EDTA Disodium — — — — 0.05 0.05 Mannitol — —— — 0.05 0.05 Hydroxyethylcellulose — — — — — 1.25 250 HHX Purifiedwater q.s. 100 q.s. 100 q.s. 100 q.s. 100 q.s. 100 q.s. 100 No BufferBuffer Buffer Buffer Buffer Buffer pH 5 pH 6 pH 7 pH 6 pH 6

TABLE 5 Stability Results of Formula 2414-14 Containing Pluronic 127 perStudy #1062 Time Physical Chemical Assay (%, LC) Storage PointProperties and Methyl- Propyl- Condition (Month) Appearance API parabenparaben Initial Clear and 98.8 92.0 96.6 colorless gel Freeze/ThawConforms 89.8 88.6 94.0  5° C. 1 Conforms 84.6 93.0 97.9 25° C. ½Conforms 90.6 93.6 98.6 1 Conforms 82.8 88.8 94.0

TABLE 6 Stability Results of Formula 2414-15 Containing HEC per Study#1062 Time Physical Chemical Assay (%, LC) Storage Point Properties andMethyl- Propyl- Condition (Month) Appearance API paraben paraben InitialClear 101.9 97 103.1 colorless gel Freeze/Thaw Conforms 98.8 97.1 103.9 5° C. 1 Conforms 96 98.1 104.6 3 Conforms 99.7 98.3 101.8 25° C. ½Conforms 91.6 99.7 106.7 1 Conforms 93.4 96.8 103 3 Conforms 85.7 92.896.5

Example 2 R&D Study Per Protocol #1074

Based on the results from study #1062, additional prototype formulationsbased on formula 2414-15 were designed and evaluated per protocol #1074.During this study, 7 prototype formulations were prepared. The purposesof the study were to select a proper buffering system and antioxidantsfor the formulation. Five (5) antioxidants were evaluated: Mannitol,Methionine, Acetylcysteine, Lysine and Histidine. Two buffer systemswere used: phosphate buffer at pH 6 and citrate buffer at pH 5. Glycerin(5%) was introduced into the new prototype formulations to provide somehumectant property to the wound treatment topical gel. PEG 200 was alsoevaluated for its potential stabilizer effect. The compositions of theprototypes evaluated per study #1074 are listed in Table 7.

TABLE 7 Composition of Prototype Formulations per Study #1074 Formula #2414- Component (% w/w) 18 19 20 21 22 23 26 Peptide 328967 (ACT1) 0.0360.036 0.036 0.036 0.036 0.036 0.036 Propylene Glycol 1.0 1.0 1.0 1.0 1.0— — PEG 200 — — — — — 5.0 5.0 Glycerin 5.0 5.0 5.0 5.0 5.0 5.0 5.0Monobasic Sodium 0.263 0.263 0.263 0.263 0.526 — — Phosphate DibasicSodium 0.044 0.044 0.044 0.044 0.088 — — Phosphate Citric Acid,anhydrous — — — — — 0.60 0.60 Sodium Citrate, — — — — — 1.73 1.73anhydrous Methylparaben 0.17 0.17 0.17 0.17 0.17 0.17 0.17 Propylparaben0.02 0.02 0.02 0.02 0.02 0.02 0.02 EDTA disodium 0.05 0.05 0.05 0.050.05 0.05 0.05 Mannitol 0.05 — — — — 0.05 — Methionine — 0.5 0.5 0.5 0.5— — Acetylcysteine — — — — 0.2 — — Lysine — — 0.1 — — — 0.1 Histidine —— — 0.1 — — — Hydroxyethylcellulose 1.25 1.25 1.25 1.25 1.25 1.25 1.25250 HHX Purified Water, qsad 100 100 100 100 100 100 100 25 mM 25 mM 25mM 25 mM 50 mM 100 mM 100 mM Buffer Buffer Buffer Buffer Buffer Buffer

Due to API availability limitations, 0.036% of API concentration wasused for this study. Formula 2414-18 is identical to 2414-15 except thatthe API concentration was reduced from 0.072% to 0.036%. The availablestability data from study #1074 found in Table 8 indicated the followingconclusions.

Phosphate buffer seemed to provide better stability to the peptide whencompared to the citrate buffer. The buffer capacity at 25 mM is adequatefor the system.

Mannitol seemed to provide adequate stability to the peptide.

The effect of other antioxidants was not clear due to the variability inthe chemical data.

Based on the study results mainly from study #1062 and partially from#1074, a final formulation was selected for further development. Thefinal formulation contained 25 mM phosphate buffer to maintain the pH ofthe formulation, mannitol as a stabilizer for the API, and HEC as agelling agent. The amount of propylene glycol was increased from 1% to3% in prototype formulations to allow for better solubilization of theparabens in the aqueous system. Four potential API concentrations wereselected: 0.0072%, 0.018%, 0.036% and 0.072%. The final formula issummarized in Table 9. The stability of the final selected formulacontaining different levels of API was also evaluated with analyticalmethods. In addition, the overall stability was assessed by visualinspection for white powder or precipitate and determination of watercontent.

TABLE 8 Stability Results of Formulas 2414-18, 2414-19, 2414-20,2414-21, 2414-22, 2414-23, and 2414-26 from Study #1074 Physical StorageTime Point Properties Chemical Assay (%, LC) Sample Condition (Month)Appearance API Methylparaben Propylparaben Formula Initial Clear and106.4 94.6 102.2 2414-18 Colorless Gel  5° C. 1 Conforms 90.1 83.4 88.22 Conforms NA NA NA 3  Conforms¹ NA NA NA 25° C. 1 Conforms 94.3 90.495.5 2 Conforms NA NA NA 3  Conforms¹ NA NA NA Formula Initial Clear and109.3 94.2 119.7 2414-19 Colorless Gel  5° C. 1 Conforms 101.1 90.4112.6 2 Conforms NA NA NA 3 Conforms¹ NA NA NA 25° C. 1 Conforms 103.394.6 117.4 2 Conforms NA NA NA 3  Conforms¹ NA NA NA Formula InitialClear and 107.0 96.9 99.9 2414-20 Colorless Gel  5° C. 1 Conforms 106.896.7 98.4 2 Conforms NA NA NA 3  Conforms¹ NA NA NA 25° C. 1 Conforms97.9 94.2 95.6 2 Conforms NA NA NA 3  Conforms¹ NA NA NA Formula InitialClear and 104.3 95.1 110.4 2414-21 Colorless Gel  5° C. 1 Conforms 96.791.5 104.2 2 Conforms NA NA NA 3  Conforms¹ NA NA NA 25° C. 1 Conforms96.4 95.0 108.4 2 Conforms NA NA NA 3  Conforms¹ NA NA NA FormulaInitial Clear and 112.2 96.1 109.4 2414-22 Colorless Gel  5° C. 1Conforms 111.4 94.9 106.1 2 Conforms NA NA NA 3 Conforms¹ NA NA NA 25°C. 1 Conforms 96.5 89.5 100.0 2 Conforms NA NA NA 3  Conforms¹ NA NA NAFormula Initial Clear and 87.8 94.7 101.6 2414-23 Colorless Gel  5° C. 1Conforms 88.3 92.9 97.0 2 Conforms NA NA NA 3  Conforms¹ NA NA NA 25° C.1 Conforms 74.4 84.4 88.4 2 Conforms NA NA NA 3  Conforms¹ NA NA NAFormula Initial Clear and 83.0 95.5 105.9 2414-26 Colorless Gel  5° C. 1Conforms 78.5 91.0 98.5 2 Conforms NA NA NA 3  Conforms¹ NA NA NA 25° C.1 Conforms 77.4 93.3 100.7 2 Conforms NA NA NA 3  Conforms¹ NA NA NA¹Particles at the bottom of vial; NA = data not available

In one embodiment, a batch of the drug product may be prepared byfollowing the steps of the manufacturing process flow chart and usingthe composition of Table 9.

Example 3 Treatment of Diabetic Foot Ulcer

A Phase II, randomized, prospective, double blind, parallel group,multi-center study was conducted to assess the safety and efficacy ofACT1 peptide in the treatment of diabetic foot ulcer.

Subjects in Group A received 100 μM of the ACT1 peptide formulation(i.e., Granexin™ Gel) in addition to standard-of-care treatment (SoC)compared to SoC alone in Group B. The total study duration for efficacyassessments was 12 weeks with additional 12 weeks follow up for safetyevaluations. The study procedures were divided into three phases:Screening Procedures (Study Days: −7 and 0), Treatment Procedures (StudyDay 0 through Study Week 12), and Follow-up Procedures (Study Months 4through 6).

Subjects enrolled in the study may have had multiple ulcers but only oneulcer was considered as the target ulcer which was within the range of4-1250 mm² and was separated from other ulcers by at least 20 mm.According to the randomization schedule, subjects had their ulcer sitetreated with either Granexin™ Gel and SoC or SoC alone. Each subject inGroup A was treated with up to 14 applications of Granexin™ Gel over theinitial 12 weeks of the study period. Other than the application ofGranexin™ Gel, management of the wound sites was equivalent in both thegroups. Each subject was assessed weekly for the first 12 weeks oftreatment. Statistical analysis of subject data up to 12 weeks was donefor efficacy and safety analyses. Wound photographs were used to assesswound closure for both control and test sites. Safety was determined byTreatment Emergent Adverse Events (TEAEs).

Inclusion Criteria:

A subject was enrolled in this study if he/she met the followingcriteria:

-   1. Male or female aged 18 years and older-   2a. Female subjects were post-menopausal or surgically sterilized,    or-   2b. Female of child-bearing potential must have had a negative    pregnancy test at Screening, and agreed to use hormonal    contraceptive or intra-uterine device or diaphragm with spermicide    or condom with spermicide or abstinence throughout the study-   3. Diabetes mellitus (type I or II) with an HbA1c<10.0%-   4. Diagnosis of neuropathic foot ulcer-   5. Cutaneous, full thickness (University of Texas grade A1), below    ankle surface ulcer between 0.5 cm² and 40 cm² post debridement-   6. A viable, granulating wound as per Investigator's discretion-   7. Ulcer present for at least 4 weeks prior to Screening-   8. An Ankle Brachial Pressure Index (ABPI) between 0.70 and 1.3    measured at Screening-   9. Signed informed consent form

Exclusion Criteria:

-   1. A subject was not eligible for this study if he/she met any of    the following criteria:-   2. Decrease or increase in the ulcer size by 30% or more during 7    day screening period-   3. Cannot tolerate the off-loading method or comply with SoC-   4. An ulcer which showed signs of severe clinical infection, defined    as pus oozing from the ulcer site-   5. The ulcer to be treated required operative debridement-   6. An ulcer positive for β-hemolytic streptococcus upon culture-   7. Requirement for total contact casts-   8. The ulcer had more than 50% slough, significant necrotic tissue,    bone, tendon, or capsule exposure-   9. Highly exuding wounds (wounds that require a daily dressing    change) ABPI<0.7 or >1.3 or ankle systolic pressure <70 mm H-   10. gHad systemic infections-   11. Met one of the following (only 1 out of 3 tests was required):-   a. Doppler waveform analysis on the dorsalis pedis and posterior    tibial arteries a monophasic or biphasic flow (with loss of reverse    flow) in either foot artery, or-   b. A toe: brachial index <0.7 or >1.3, or-   c. Transcutaneous oxygen pressure <40 mm Hg-   12. Presence of an active systemic or local cancer or tumor of any    kind (with the exception of non-melanoma skin cancer)-   13. Congestive Heart Failure New York Heart Association class II-IV    or coronary heart disease with ST segment elevation myocardial    infarction or coronary artery bypass graft or percutaneous    transluminal coronary angioplasty within the last 6 months-   14. Active osteomyelitis of the study foot-   15. Active connective tissue disease-   16. Acute Charcot's neuro-arthropathy as determined by clinical    and/or radiographic examination-   17. Active treatment with systemic corticosteroids-   18. Previous or current radiation therapy to the distal lower    extremity or likelihood to receive this therapy during study    participation-   19. Pregnant or nursing subjects-   20. Uncontrolled anemia (Hb<10 g/dL in females and <12 g/dL in    males)-   21. Estimated glomerular filtration rate <25 mL/min-   22. Poor nutritional status defined as an albumin <25 g/L-   23. Significant peripheral edema as per Investigator's discretion-   24. Known prior inability or unavailability to complete required    study visits during study participation-   25. A psychiatric condition (e.g. suicidal ideation) or chronic    alcohol or drug abuse problem, determined from the subject's medical    history, which, in the opinion of the Investigator, may pose a    threat to subject compliance-   26. Use of a platelet-derived growth factor within 28 days prior to    Screening-   27. Use of any investigational drug or therapy within 28 days prior    to Screening-   28. Any other factor which may have, in the opinion of the    Investigator, compromised participation and follow-up in this study

Granexin™ Gel (100 μM ACT1 peptide, in the formulation described inTable 1 of this application) was applied topically on the wound on Day0, Day 3, Week 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12.

Total duration of the study was 12 months, including 6 months enrollmentperiod. Individual subject participation for the period of interimanalysis was approximately 12 weeks.

Efficacy:

Efficacy was evaluated by assessment of the following parameters betweenstudy Day 0 to Week 12:

-   -   Wound photographs*    -   Wound tracings**    -   Wound closure assessment*    -   Wound characteristics (clinical assessment)**    -   Wound treatment response**

Safety:

The safety was evaluated by assessing the following parameters:

-   -   TEAEs (each visit from Day 0 up to Week 12)*    -   Vitals (Day −7 and Week 12 or Last Subject Last Visit [LSLV])**    -   Laboratory assessments (Day −7 and Week 12 or LSLV)**

* These evaluations were considered for the interim analysis

** These evaluations were not considered for the interim analysis

Sample Size Calculations

The sample size was calculated with reference to the primary endpoint(comparing the reduction in wound size as measured by the meanpercentage decrease from Baseline to the last visit). Assuming a 25%difference in favor of subjects treated with Granexin™ Gel, power of80%, significance level of 95% (two-sided) and Standard Deviation (SD)of 40%, 40 subjects were calculated to be required in each treatmentgroup. Adjusting for 15% dropout rate, 92 subjects were required to beenrolled in the study.

All statistical tests were carried out as two-sided on 5% level ofsignificance unless otherwise stated. Summary tables and descriptivestatistics were done for demographics, efficacy, and safety variables.To adjust the main efficacy analyses for measured covariates atBaseline, Analysis of Co-Variance (ANCOVA) and survival analysis wereperformed. The descriptive statistics for continuous variables werepresented with number (N) of non-missing observations, mean, SD, median,minimum and maximum or range. For categorical data, descriptivestatistics were presented with number of exposed subjects and number (N)with percentage of observations in the various categories of theendpoint, where percentage was based on the exposed subjects.

Primary efficacy analysis was performed on Intent-to-Treat (ITT) and PerProtocol (PP) populations. Primary efficacy data was calculated usingsample number (n), mean, SD, median, minimum, maximum, and 95%Confidence Interval (CI).

ANCOVA with repeated measures was performed for comparison of meanpercent reduction of wound area from Baseline compared to Week 12between the two treatment groups. Mean percent reduction of wound areafrom Baseline to Week 12 was considered as response variable adjustedfor the strata, wound duration, viable tissue, exudate level, and theBody Mass Index (BMI) as covariates and treatment group, treatment byvisit interaction, and visit as factor included in ANCOVA model.

The mean percent wound closure at 4 weeks compared to Baseline wasanalyzed as described in the primary efficacy analysis. Subjectassessment of intensity of pain at Week 12 was analyzed by the WilcoxonMann-Whitney U test.

For time-to-event endpoints, the distribution for each treatment groupwas estimated by the Kaplan-Meier method and compared by the log-ranktest. The median time along with 90% confidence limits was presented foreach treatment group. Simultaneous confidence bounds for theKaplan-Meier curve were computed for both the treatment groups. Further,Cox proportional hazard regression model analysis was conducted tocompare the event-time distribution function between the treatmentgroups. The incidence of 100% study wound closure was a time-to-eventanalysis computed using Kaplan-Meier methods where all scheduled visitsfrom Day 0 though Week 12 were included in the analysis. This was theprimary analysis of the secondary efficacy endpoint. Summary statistics,including the median for incidence of 100% study wound closure waspresented. The statistical significance of the difference in theincidence of 100% wound closure was evaluated using the log-rank test.Cox's proportional hazard regression analysis was performed to evaluatethe individual and joint effects of several covariates on the incidenceof 100% wound closure.

Incidence of 100% wound closure (closed or not closed) by or on Week 12was analyzed overall and by center. Statistical significance infrequency between treatment groups were assessed by Fisher's Exact test(two-tailed) to determine the overall wound healing frequencies. Thiswas followed by the Cochran-Mantel-Haenszel test which compared the 2treatment groups with respect to the incidence of 100% wound closureafter adjusting for pooled center. The Breslow-Day test was used inconjunction with the Cochran-Mantel-Haenszel test to determine thestatistical significance of a treatment by-pooled-center interaction

Results

Mean Percent Wound Closure from Baseline to Week 12

The primary efficacy endpoint of the study was to evaluate mean percentwound closure from Baseline to Week 12. The non-parametric WilcoxonMann-Whitney U test was used to analyze mean percent reduction of woundarea from Baseline to Week 12 in both the treatment groups since thedata did not follow a normal distribution, as confirmed by aShapiro-Wilk p-value of <0.0001 and a non-linear Q-Q plot. The additionof the ranks of both groups obtained after sorting the data of woundsize reduction in ascending order in the PP population showed that theactual sum of scores (1036.00) was higher in Group A subjects than theexpected sum of scores i.e. sum of scores when there is no difference in2 groups, under the null hypothesis (899.00) and in Group B, the actualsum of scores (617.00) was found to be lower than the expected sum ofthe scores under the null hypothesis (754.00). Therefore, in the PPpopulation, subjects treated with Granexin™ Gel plus SoC (Group A) had ahigher percent wound closure from Baseline to Week 12 compared tosubjects treated with SoC alone (Group B), which was statisticallysignificantly (p-value=0.0069).

Mean Percent Wound Closure from Baseline to Week 4

Similarly non-parametric analysis for secondary endpoint showed that inthe PP population, the mean percent reduction of wound area fromBaseline to Week 4 was statistically significantly higher in Group A ascompared to Group B (p-value=0.0128).

Subject Self Assessment of Change in Intensity of Pain from Baseline toWeek 12

The intensity of pain experienced by the subjects was recorded on aVisual Analogue Scale of 1 to 10 where 1 indicated “no pain” and 10indicated “extreme pain”. The intensity of pain as assessed by thesubjects of the PP population at treatment Week 12 of the study showedusing the Wilcoxon Mann-Whitney U test that there was no statisticallysignificant difference in subject self assessment of intensity of painat Week 12 between the 2 treatment groups (Group A, 0.4±1.22; Group B,0.3±0.81; p-value=0.9683).

Time-to-Wound Closure Analysis

The time taken for complete wound closure in the PP population wasstatistically significantly lower in subjects in Group A as compared tosubjects in Group B (p-value=0.0057). Of the 32 subjects in Group A,81.3% subjects had complete (100%) wound closure and 18.8% subjects whodid not, were censored. The median duration of 100% wound closure inGroup A was 6 weeks. Of the 28 subjects in Group B, 53.6% subjects hadcomplete wound closure and 46.4% subjects not having 100% wound closure,were censored and the median duration of time to complete 100% woundclosure was 14.64 weeks.

The time taken to achieve 50% wound closure by Week 12 in the PPpopulation was not statistically significantly lower in subjects inGroup A as compared to the time taken by subjects in Group B(p-value=0.2084). The analysis of time taken for complete 50% woundclosure by Week 12 showed that of the 32 subjects in Group A, 93.8%subjects completed 50% wound closure and 6.3% subjects did not complete50% wound closure and were censored. In comparison to that, out of 28subjects in Group B, 89.3% subjects had 50% wound closure and 10.7%subjects without at least 50% wound closure during the study period werecensored. The median duration of 50% wound closure was 2 weeks in GroupA and 4.07 weeks in Group B.

Cox Proportional Hazard Regression Analysis for time to 100% woundclosure in the PP population showed that though treatment was not astatistically significant factor affecting the wound closure(p-value=0.0856), the subjects in Group A treated with Granexin™ Gelplus SoC would have wound closure 2.339 times more than the subjects inGroup B treated with SoC only. Similarly, baseline wound depth and BMIwere not statistically significant factors (baseline wound depth:p-value=0.4554, BMI: p-value=0.4435) but would affect wound closure by1.715 times and 1.050 times, respectively. All other parameters,including wound size, wound duration, and use of dressings did not havea statistically significant effect on the rate of wound closure for boththe treatments.

Incidence of 100% Wound Closure by or on Week 12

The categorical analysis of incidence of 100% and 50% wound closure byor on Week 12 in the PP population was done using theCochran-Mantel-Haenszel analysis with Breslow-Day test and theChi-square test. The Chi-square test performed by combining the datafrom all the study centers showed that there were statisticallysignificantly higher number of responders in Group A than in Group B(100% wound closure: p-value=0.0104; 50% wound closure: p-value=0.0063).Therefore it can be concluded that the frequency of 100% and 50%epithelialization occurred at a higher frequency in wounds of subjectstreated with Granexin™ Gel than subjects treated with SoC only.

Safety:

Overall, 21 subjects reported 26 AEs of which 18 (69.2%) AEs were mildin intensity. A total of 4 moderate and 4 severe AEs were reported inthis study. In Group A, death and foot fracture were the severe AEs andmyocardial infarction and inadequate control of diabetes mellitus weresevere AEs reported in Group B. None of the AEs reported in this studywere considered probably or possibly related to Granexin™ Gel or the SoCtreatment. Of the 26 events, 18 (69.2%) recovered and 3 (11.5%) eventsrecovered with sequelae. No action was taken against 22 AEs. None of thesubjects withdrew due to an AE. Five subjects reported 5 Serious AdverseEvents (SAEs). In Group A, 1 death was reported due to an unknown causeand 1 death in Group B occurred due to myocardial infarction. None ofthe SAEs, including the deaths, were related to Granexin™ Gel.

This phase II study was a double blind, randomized, prospective,parallel group, multi-center study conducted at 8 centers in India toevaluate the efficacy and safety of Granexin™ Gel in the treatment ofDFU. The total duration of the study was 12 months, including 6 monthsenrollment period and 6 months for study procedures (3 months each forefficacy and safety analyses). Hence, the study duration for anindividual subject was approximately 6 months, including 18 visits. Forinterim analysis, data up to 12 weeks (Visit 15) was considered. A totalof 92 subjects were randomized in the study to receive Granexin™ Gelplus SoC or only SoC in the treatment of DFU. Of these subjects, 76(82.6%) completed the study treatment visits up to Visit 15 and are nowin the follow-up stage of the study.

The primary efficacy endpoint of the study was to evaluate mean percentwound reduction from Baseline to Week 12. The non-parametric WilcoxonMann-Whitney U test was used to analyze mean percent reduction of woundarea from Baseline to Week 12. In the PP population, subjects treatedwith Granexin™ Gel plus SoC (Group A) showed statistically significantlyhigher percent wound closure from Baseline to Week 12 compared tosubjects treated with SoC alone (Group B) (p-value=0.0069), suggestingthat at Week 12 wounds treated with Granexin™ Gel plus SoC healed betterthan the wounds treated with SoC alone.

The secondary endpoints of the study were to assess mean percent woundclosure at Week 4, subject assessment of pain, and time to completewound closure. At Week 4, Group A subjects in the PP population showedstatistically significantly higher percent reduction of wound area fromBaseline compared to Group B subjects in the PP population. There was nostatistically significant difference in intensity of pain as assessed bythe subject from Baseline to Week 12 between the 2 treatment groups.Hence, Granexin™ Gel did not affect the intensity of pain experienced bythe subjects as recorded on a Visual Analogue Scale of 1 to 10.

Complete wound closure was defined as 100% epithelialization of thewound and the absence of drainage from the wound. In the PP population,subjects in Group A took significantly less time to achieve 100% woundclosure than those in Group B. However, the time taken for 50% woundclosure was not significantly lower in Group A as compared to Group B.The categorical analysis of incidence of 100% and 50% wound closure atWeek 12 was conducted and the subjects who had 100% wound closure wereconsidered as responders. The number of responders in Group A wassignificantly higher than in Group B, indicating that the wounds treatedwith Granexin™ Gel plus SoC have higher incidences of 100% and 50%epithelialization than wounds treated with SoC alone.

Cox Proportional Hazard Regression Model was used to compare theevent-time distribution function between the treatment groups. Althoughtreatment was not a significant factor affecting time to 100% woundclosure, in the PP population Group A subjects demonstrated 2.3 timeshigher wound closure than Group B subjects. In addition, baseline wounddepth and BMI were not significant factors but affected wound closure by1.715 times and 1.050 times, respectively. All other parameters,including wound size, wound duration, and use of dressings did not havea statistically significant difference in affecting wound closure forboth the treatments. Cox Proportional Hazard Regression Analysis fortime to 50% wound closure showed that none of the covariates (treatmentgroup, wound size, wound duration, baseline wound depth, and BMI) weresignificantly associated with time to 50% wound closure. However, GroupA subjects had wound closure 1.186 times more than Group B subjects.

In terms of safety, out of 92 subjects, 21 (22.8%) subjects reported 26AEs. There was no significant difference in the number of subjects withat least 1 AE between the 2 treatment groups (Group A, 11; Group B, 10;p-value=0.8038). A total of 4 AEs were of severe intensity. None of theAEs were related to the Granexin™ Gel or SoC. None of the subjectswithdrew due to an AE. A total 5 (5.4%) subjects reported 5 SAEs where 2SAEs were observed in Group A subjects and 3 SAEs were in Group Bsubjects. In Group A, 1 death was reported due to an unknown cause and 1death in Group B occurred due to myocardial infarction. None of the SAEswere related to Granexin™ Gel or SoC.

In conclusion, Granexin™ Gel along with SoC has shown to acceleratewound healing in subjects with DFU, making it an efficacious, safe, andwell tolerated therapeutic option for the treatment of DFU.

The description of 18 visits along with premature study discontinuationand unscheduled visits is summarized below:

Visit 1: Screening (Day −7)

The Screening Visit was held a maximum of 7 days prior to the BaselineVisit. The following procedures were performed at the Screening Visit:Screening Procedures: informed consent, medical history, physicalexamination including neuro-vascular examination, vitals, ulceridentification and history, assessment of wound infection, X-rays,Electrocardiogram (ECG), and serum pregnancy testInclusion and exclusion criteria checkedLaboratory testsImmunology testsPhotography of the ulcerWound debridementWound tracingWound bed preparationRecorded the use of concomitant medicationRecorded the presence of concomitant illness(es)

Visit 2: Baseline (Day 0)

The Baseline Visit was held a maximum of 7 days after Screening. Ifulcers healed by >30% within 7 days of the start of the study at Day 0,then the subject was not eligible for participation in the study. Thefollowing procedures were performed at Baseline:Inclusion and exclusion criteria checked to confirm that no change hadoccurred since Screening

Randomization

Photography of the ulcerWound tracingClinical assessmentCompliance checkedWound closure assessmentWound bed preparationTreatment with Granexin™ Gel (if applicable)Dressing changedPain assessmentRecorded changes in concomitant medication

Recorded Adverse Events (AEs), if any

Visit 3-14: Treatment Visits (Day 3 Through Week 11)

On Treatment Visits were conducted at Day 3 followed by Week 1, 2, 3, 4,5, 6, 7, 8, 9, 10, and 11 after Baseline Visit ±1 day. The followingprocedures were performed at these visits:Photography of the ulcerWound tracingClinical assessmentCompliance checkedWound closure assessmentWound bed preparationTreatment with Granexin™ Gel (if applicable)Dressing changedPain assessmentRecorded changes in concomitant medication

Recorded AEs, if any

Visit 15: End of Treatment Visit (Week 12)

The End of Treatment Visit was conducted at Visit 15 (Week 12±1 day).The following procedures were performed at this visit:Laboratory testsImmunology testsPhotography of the ulcerWound tracingClinical assessmentCompliance checkedWound closure assessmentWound bed preparationTreatment with Granexin™ Gel (if applicable)Dressing changedPain assessmentRecorded changes in concomitant medicationAdditional end of treatment visit evaluations (assessment of ulcerresponse to treatment, comparison of current treatment to previoustreatment, incidence of ulcer recurrence within the 12-week treatmentperiod, and vital signs)

Recorded AEs, if any

Visit 16-18: Follow-Up Visits, Including End of Study Visit (Month 4Through 6)

The Follow-up visits would be conducted at Month 4±3 days, Month 5±3days, and Month 6±3 days (End of Study [EOS] visit). The followingprocedures would be performed at these visits:Photography of the ulcerPain assessmentRecorded changes in concomitant medicationFollow-up procedures (evaluation of ulcer recurrence, care of unhealedulcers, assessment of healed wounds, and subject assessment of pain)

Recorded AEs, if any

EOS procedures (only at Visit 18)

Granexin™ Gel is a topical hydrogel that contains the active ingredientACT1 peptide (100 μM). Granexin™ Gel has greater than 90% water, whichprovides a barrier to infection or external elements while maintaining amoist environment for the wound. In Group A, the ulcer was cleansed withsterile saline and if any bleeding existed, it was stopped beforeapplying the Granexin™ Gel on Days 0 and 3, and Weeks 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, and 12. The Granexin™ Gel was dispensed directly fromits 5 ml glass vial(s) to completely cover the wound bed. With a cleansterile cotton swab, sterile tongue depressor, or similar applicationaid, the Granexin™ Gel was evenly spread over the wound surface. Thetreatment site was then dressed according to the following regimen:

Used adequate off-loading other than TCC as per the Investigator'sjudgment

Applied a non-adherent dressing over the ulcer and Granexin™ Gel whichextended 0.5 inch beyond the ulcer perimeter and inflamed skin margins

Applied a non-occlusive dressing such as fine mesh gauze which waseither folded or rolled as a bolster

Standard of Care (Control) Treatment

In the control group, the ulcer was cleansed with sterile saline and ifany bleeding existed, it was stopped before dressing. The ulcer wasdressed according to the following regimen:

Used adequate off-loading other than TCC as per the Investigator'sjudgment

Applied a non-adherent dressing such as the 4 layer dressing over theulcer with either soframycin, Lyseal ointment or hydroheal AM gel whichextended 0.5 inch beyond the ulcer perimeter and inflamed skin margins

Applied a non-occlusive dressing such as pressure bandage or fine meshgauze which was either folded or rolled as a bolster

Granexin™ Gel is a topical gel with the active drug substance ACT1peptide developed at FirstString Research for the management of wounds.ACT1 is a synthetic peptide (25 amino acid) designed to mimic theCarboxy (C)-terminus of the ubiquitous transmembrane gap junctionprotein Connexin 43 (Cx43), with high binding specificity to zonaoccludens, a cytoplasmic tight junction protein. While the mechanism ofaction of ACT1 has not been completely elucidated, the peptide issoluble and engineered to directly translocate within cells. ACT1peptide interacts with a known binding partner of Cx43—the PostsynapticDensity 95/Discs Large/Zonula Occludens (PDZ)-2 domain of ZonulaOccludens-1 (ZO-1). The binding of the peptide to specific Cx43C-terminus interaction domains such as PDZ-2 on ZO-1 serves tocompetitively inhibit its association with the Cx43 C-terminus.Connexins are gap junction proteins where as ZO-1 is a tight junctionassociated protein. The binding of the 25 amino acid ACT1 peptide to thePDZ-2 domain of ZO-1 serves to stabilize gap junctions as well as tightjunctions. This junctional stability has been associated with fasterhealing and reduced scarring. In addition, the low molecular weight (25amino acid) peptide is expected to reduce the risk of an immunogenicresponse.

Granexin™ Gel is also designed to provide a localized protective barrieragainst microbial colonization and a moist environment to promotenatural autolytic debridement of necrotic tissue surrounding the wound.The lot number of Granexin™ Gel used in this study was DP 1493.

Subjects were randomized 1:1 to either Group A (Granexin™ Gel plus SoC)or Group B (SoC only). Interactive Web Response System (IWRS) was usedfor randomization of the eligible subjects to the treatment group. Therandomization was further stratified by wound size (<10 cm² and >10 cm²)to avoid bias of undue number of subjects with small or large woundsgoing into one arm of the study. The subject ID, initials and study Day0 date were written on the randomization code sheet and filed with thesubject's CRF. Randomization lists were prepared centrally by the MNIdata management center using a validated computer program (StatisticalAnalysis Software [SAS] 9.1.3).

Two studies, including a preclinical and clinical study tested theefficacy and safety of 100 μM concentration of ACT1 formulated inGranexin™ Gel. In the preclinical study, the efficacy of Granexin™ Gelin wound healing was assessed on 5 mm diameter full thickness excisionalwounds in adult mice and pigs. Wounds treated with 100 μM of ACT1peptide closed faster, appeared less swollen and inflamed, and healedwith a smoother and less discolored appearance as compared to the woundsapplied with the vehicle control [18]. Subsequently, a phase I, doubleblind, single-center, controlled study was conducted to evaluate thesafety and tolerability of 20, 50, 100, and 200 μM concentrations ofACT1 formulated in Granexin™ Gel versus placebo in 48 healthy subjectsfollowing punch biopsy. The results showed the Granexin™ Gel (ACT1) ateach concentration to be safe and well tolerated with no evidence oflocalized or systemic AEs or Serious Adverse Events (SAEs). Bothpreclinical and clinical studies concluded 100 μM concentration of theACT1 peptide to be optimum in safety and effectiveness. Hence, the doseof 100 μM ACT1 of Granexin™ Gel was selected in the treatment of DFU inthis study.

For each subject, Granexin™ Gel (100 μM ACT1) was applied at Days 0 and3, and Weeks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12

Efficacy and Safety Variables:

The primary efficacy variable was mean percent wound closure fromBaseline to Week 12

Secondary Efficacy Variables

-   -   Mean percent wound closure at 4 weeks    -   Subject self-assessment of intensity of pain (till Week 12)    -   Time to 50% wound closure    -   Time to complete wound closure with 100% epithelialization of        the wound

Safety Variable:

The safety variable was incidence of treatment related Adverse Events(AEs) (till Week 12).

An AE was defined as “any untoward medical occurrence in a subject orclinical investigation subject administered a pharmaceutical productthat did not necessarily have a causal relationship with this treatment”(ICH E6:1.2). An AE, therefore, was any unfavorable and unintended sign,symptom, or disease temporally associated with the use of a medicinalproduct, whether or not considered related to the medicinal product.

A clinically significant abnormal laboratory finding was also regardedas an AE and it was the responsibility of the Investigator to review thelaboratory test results and determine whether an abnormal laboratoryvalue was of clinical significance. In general, clinically significantlaboratory values which suggested disease progression and/or requiredactive management were considered as AEs.

Worsening of a pre-existing medical condition (e.g. diabetes, migraine,headaches, and gout) was considered as an AE if there was either anincrease in severity, frequency, duration of the condition, or anassociation with significantly worse outcomes. Pre-planned proceduresand pre-existing conditions that did not worsen were not considered AEs.

A Serious Adverse Event (SAE) was defined as an AE that:

Was fatalWas life threatening (places the subject at immediate risk of death)Required in-subject hospitalization or prolongation of existinghospitalizationResulted in persistent or significant disability/incapacityWas a congenital anomaly/birth defectMet other significant medical hazard

An AE necessitating hospitalization met the regulatory definition for“serious” if the in-subject hospital admission included a minimum of anovernight stay in a health care facility. Any AE that did not meet oneof the definitions of serious (e.g. an AE requiring an emergency roomvisit, out-subject surgery, or requiring urgent investigation) wasconsidered by the Investigator to meet the “other significant medicalhazard” criterion for classification as an SAE.

If the above interventions were performed as SoC and not associated withan AE, the health issue for which the intervention was performed was notconsidered as an SAE. If there was a complication as a result of theprocedure and the complication met at least 1 seriousness criterion,then that complication was reported as an SAE.

Study Subjects: A total of 98 subjects with DFU were screened. Of these,6 were screen failures. The remaining 92 subjects were randomized in thestudy. Of these subjects, 76 (82.6%) completed the study treatmentvisits up to Visit 15 and are now in follow-up. The remaining 16subjects did not complete the study treatment visits up to Visit 15(Table 9).

TABLE 9 Disposition of Subjects Treatment Group Group A Group B OverallCategory (N = 46) (N = 46) (N = 92) Population, n (%) ^([1]) Screened 98Randomized 46 (100.0)  46 (100.0)  92 (100.0) Intent-to-treat 46 (100.0)45 (97.8) 91 (98.9) Per-Protocol 32 (69.6)  28 (60.9) 60 (65.2) Totalnumber of subjects 37 (80.4)  39 (84.8) 76 (82.6) completed the study(Visit 15) Total number of subjects not 9 (19.6)  7 (15.2) 16 (17.4)completed the study (Visit 15) Note: ^([1]) Percentage was calculatedtaking respective column header group count as denominator.

The following were the reasons, which led to the exclusion of 32subjects from the PP population: Consent withdrawal (13 subjects); Lostto follow up (2 subjects); Death (2 subjects); Drop outs (1 subject);Protocol deviation(s); Decrease or increase in the ulcer size by 30% ormore during 7 day screening period (14 subjects)

ITT Population:

All subjects randomized to any treatment group, received at least onedose of study medication or reference treatment and performed studyassessments within and/or outside the time window period specified inthe protocol. The assessments performed outside time window period werere-assigned to the closest nominal time point of actual samplecollection

PP Analysis Population:

All subjects who completed study assessments within the protocol definedwindow period for the assigned randomized time point and also completedall study assessments until Week 12. This included subjects who came forall the visits within or outside the window period, including subjectswhose wounds had healed before Week 12. Subjects who had a majorprotocol deviation were excluded from the PP population. This includedsubjects who had an increase or decrease of more than 30% in wound sizeduring the screening period of 7 days (per exclusion criteria no. 1 inthe protocol)

Safety Population:

All subjects who were randomized in the study to either of the treatmentgroups were used for the safety analysis

The ITT population included 91 subjects; the PP population included 60subjects; and the safety population included 92 subjects.

Primary Efficacy Endpoint:

Mean Percent Wound Closure from Baseline to Week 12

Parametric Analysis: Table 10 summarizes the parametric analysis of themean percent reduction of wound area from Baseline to Week 12 in the ITTpopulation. Of 91 subjects in the ITT population, non-missingobservations for change from Baseline to Week 12 for percent reductionof wound area were available for 63 subjects. The mean percent±SDreduction of wound area from Baseline to Week 12 was higher in Group Athan Group B (Group A: 72.12%±128.52; Group B: 57.09%±80.88) but was notstatistically significant (p-value=0.6567) as per parametric analysis.

TABLE 10 Mean Percent Reduction of Wound Area from Baseline to Week 12(Parametric analysis) ITT Population (N = 91) Treatment Group Group AGroup B Statistics (N = 46) (N = 45) Total number of subjects evaluatedN = 63^([1]) Percent reduction of wound area from Baseline to Week 12 n34 29 Mean 72.121 57.086 SD 128.5222 80.8795 Median 100.000 100.000Range (Min.:Max.) (−648.91:100.00) (−271.50:100.00) LS Mean Estimate55.490 44.032 Difference Estimate ^([2]) 11.4575 SE ^([3]) 25.6968 95%CI (L.:U.) (−39.53:62.44) p-value ^([3], [5]) 0.6567 ^([1])“Total no. ofsubjects evaluated” represents subjects having non-missing observationsfor change from Baseline to Week 12 for percent reduction of wound area^([2]) Difference estimate for Granexin ™ Gel plus SoC indicated(Granexin ™ Gel plus SoC) − SoC). ^([3]) SE of Granexin ™ Gel plusStandard of Care indicated Standard Error of Differences (Granexin ™ Gelplus SoC) − SoC). ^([4]) p-value for Granexin ™ Gel plus SoC indicatedsignificance of treatment differences (Granexin ™ Gel plus SoC) − (SoC).^([5]) ANCOVA mixed model with repeated measures was used to compareaverage percent reduction in wound area with treatment, visit andtreatment*visit as factors and strata, wound duration, viable tissue,exuda to level and the BMI as covariates using PROC Mixed procedure ofSAS software.

The parametric analysis of the mean percent reduction of wound area fromBaseline to Week 12 in the PP population is summarized in Table 11. Ofthe 60 subjects in the PP population, non-missing observations forchange from Baseline to Week 12 for reduction of wound area wereavailable for 57 subjects. The mean percent±SD reduction of wound areafrom Baseline to Week 12 was statistically significantly higher in GroupA than in Group B (Group A: 93.58%±17.67; Group B: 52.14%±84.14;p-value=0.0202).

TABLE 11 Mean Percent Reduction of Wound Area from Baseline to Week 12(Parametric analysis) PP Population (N = 60) Treatment Group Group AGroup B Statistics (N = 32) (N = 28) Total number of subjects evaluatedN = 57^([1]) Percent reduction of wound area from Baseline to Week 12 n31 26 Mean 93.582 52.135 SD 17.6708 84.1427 Median 100.000 100.000 Range(Min.:Max.) (27.61:100.00) (−271.50:100.00) LS Mean Estimate 69.03830.178 Difference Estimate ^([2]) 38.8605 SE ^([3]) 16.4998 95% CI(L.:U.) (6.18:71.54) p-value ^([4],) ^([5]) 0.0202 Note: ^([1])“Totalno. of subjects evaluated” represents subjects having non-missingobservations for change from Baseline to Week 12 for percent reductionof wound area ^([2]) Difference estimate for Granexin ™ Gel plus SoCindicated (Granexin ™ Gel plus SoC) − SoC). ^([3]) SE of Granexin ™ Gelplus Standard of Care indicated Standard Error of Differences(Granexin ™ Gel plus SoC) − SoC). ^([4]) p-value for Granexin ™ Gel plusSoC indicated significance of treatment differences (Granexin ™ Gel plusSoC) − (SoC). ^([5]) ANCOVA mixed model with repeated measures was usedto compare average percent reduction in wound area with treatment, visitand treatment*visit as factors and strata, wound duration, viabletissue, exudate level and the body mass index as covariates using PROCMixed procedure of SAS software. General Note: Group A indicatesGranexin ™ Gel plus SoC and Group B indicates SoC only SD: StandardDeviation, Min: Minimum, Max: Maximum, CI: Confidence Interval, L: LowerLimit, U: Upper Limit Missing data was accounted by using repeatedmeasures analysis. Mean percent reduction from Baseline = ((Baseline −week X)/Baseline)*100

To confirm the applicability of parametric statistical inferencemethodology in the wound reduction analysis, it was tested whether thedata followed normal distribution. Using PROC UNIVARIATE of SAS®(version 9.1.3) Shapiro-Wilk W statistic was calculated. TheShapiro-Wilk p-value<0.0001 and this indicated that the data did notfollow the normal distribution. Therefore, the hypothesis of normaldistribution of the data in the sample was rejected and non-parametricanalysis was also conducted.

Table 12 summarizes the non-parametric analysis for mean percentreduction of wound area from Baseline to Week 12 in the ITT population.The Wilcoxon Mann-Whitney U test is a non-parametric test whichdetermines whether there is a difference in two samples of independentobservations. The actual sum of scores (1215.00) obtained by adding theranks of both groups after sorting the data of wound size reduction inascending order, was found higher in Group A subjects than the expectedsum of scores i.e. sum of scores when there is no difference in twogroups, under the null hypothesis (1088.00) and in Group B, the actualsum of scores (801.00) was found lower than the expected sum of thescores under the null hypothesis (928.00). Subjects in Group A hadstatistically significantly higher percent reduction of wound area fromBaseline to Week 12 than Group B subjects (p-value=0.0287).

TABLE 12 Mean Percent Reduction of Wound Area from Baseline to Week 12(Non-parametric analysis) IIT Population (N = 91) Treatment Group GroupA Group B Statistics (N = 46) (N = 45) Percent reduction of wound areafrom Baseline to Week 12 Sum of Score 1215.00 801.00 Expected Sum ofScores under H_(o) 1088.00 928.00 p-value ^([1]) 0.0287 Note: ^([1])p-value is evaluated by using non-parametric Wilcoxon Mann-Whitney Utest between treatment group. General Note: Mean percent reduction fromBaseline = ((Baseline − week X)/Baseline)*100 H_(o): Null Hypothesis

Table 13 summarizes the non-parametric analysis for mean percentreduction of wound area from Baseline to Week 12 in the PP population.The addition of the ranks of both groups obtained after sorting the dataof wound size reduction in ascending order gave the actual sum of scoresas 1036.00 which was found to be higher in Group A subjects than theexpected sum of scores (899.00) i.e. sum of scores when there is nodifference in the two groups, under the null hypothesis and in Group Bthe actual sum of scores (617.00) was found to be lower than theexpected sum of the scores under the null hypothesis (754.00). Subjectsin Group A had statistically significant higher percentage of reductionof wound area from Baseline to Week 12 than Group B subjects(p-value=0.0069).

TABLE 13 Mean Percent Reduction of Wound Area from Baseline to Week 12(Non-parametric analysis) PP Population (N = 60) Treatment Group Group AGroup B Statistics (N = 32) (N = 28) Percent reduction of wound areafrom Baseline to Week 12 Sum of Score 1036.00 617.00 Expected Sum ofScores under H_(o) 899.00 754.00 p-value ^([1]) 0.0069 ^([1]) p-value isevaluated by using non-parametric Wilcoxon Mann-Whitney U test betweentreatment group. General Note: Mean percent Reduction from Baseline =((Baseline − week X)/Baseline)* 00 H_(o): Null Hypothesis

The parametric evaluation of mean percent wound closure from Baseline toWeek 4 in the ITT population is summarized in Table 14. Of 91 subjectsin the ITT population, non-missing observations from Baseline to Week 4for percent reduction of wound area were available for 60 subjects. Themean percent±SD reduction of wound area from Baseline to Week 4 was53.30%±126.23 in Group A subjects and 52.92%±42.60 in Group B subjects;but the reduction in Group A was not statistically significant comparedto Group B (p-value=0.8234).

TABLE 14 Mean Percent Reduction of Wound Area from Baseline to Week 4(Parametric analysis) ITT Population (N = 91) Treatment Group Group AGroup B Statistics (N = 46) (N = 45) Total no. of subjects evaluated N =60^([1]) Percent reduction of wound area from Baseline to week 4 N 33 27Mean 53.306 52.920 SD 126.2293 42.6001 Median 81.414 61.484 Range(Min.:Max.) (−626.65:100.00) (−72.94:100.00) LS Mean Estimate 46.93141.098 Difference Estimate ^([2]) 5.8329 SE ^([3]) 26.0720 95% CI(L.:U.) (−45.87:57.54) p-value ^([4], [5]) 0.8234 Note: ^([1])“Total no.of subjects evaluated” represents subjects having non-missingobservations for all the dependent and covariate variables ^([2])Difference estimate for Granexin ™ Gel plus SoC indicated (Granexin ™Gel plus SoC) − SoC). ^([3]) SE of Granexin ™ Gel plus SoC indicatedStandard Error of Differences (Granexin ™ Gel plus SoC) − SoC). ^([4])p-value for Granexin ™ Gel plus SoC indicated significance of treatmentdifferences (Granexin ™ Gel plus SoC) − (SoC). ^([5]) ANCOVA mixed modelwith repeated measures was used to compare average percent reduction inwound area with treatment, visit and treatment*visit as factors andstrata, wound duration, viable tissue, exudate level and the body massindex as covariates using PROC Mixed procedure of SAS software.

The parametric analysis of mean percent reduction of wound area fromBaseline to Week 4 in the PP population is summarized in Table 15. Ofthe 60 subjects in the PP population, non-missing observations fromBaseline to Week 4 were available for a total of 49 subjects. The meanpercent±SD reduction of wound area from Baseline to Week 4 was73.49%±35.52 in Group A subjects and 47.43%±43.61 in Group B subjects;but the reduction was not statistically significantly higher in Group Asubjects than Group B (p-value=0.0871).

TABLE 15 Mean Percent Reduction of Wound Area from Baseline to Week 4(Parametric analysis) PP Population (N = 60) Treatment Group Group AGroup B Statistics (N = 32) (N = 28) Total no. of subjects evaluated N =49^([1]) Percent reduction of wound area from Baseline to week 4 N 26 23Mean 73.487 47.430 SD 35.5187 43.6110 Median 88.167 56.931 Range(Min.:Max.) (−40.60:100.00) (−72.94:100.00) LS Mean Estimate 53.18424.019 Difference Estimate ^([2]) 29.1649 SE ^([3]) 16.9134 95% CI(L.:U.) (−4.31:62.64) p-value ^([4], [5]) 0.0871 Note: ^([1])“Total no.of subjects evaluated” represents subjects having non-missingobservations for all the dependent and covariate variables ^([2])Difference estimate for Granexin ™ Gel plus SoC indicated (Granexin ™Gel plus SoC) − SoC). ^([3]) SE of Granexin ™ Gel plus SoC indicatedStandard Error of Differences (Granexin ™ Gel plus SoC) − SoC). ^([4])p-value for Granexin ™ Gel plus SoC indicated significance of treatmentdifferences (Granexin ™ Gel plus SoC) − (SoC). ^([5]) ANCOVA mixed modelwith repeated measures was used to compare average percent reduction inwound area with treatment, visit and treatment*visit as factors andstrata, wound duration, viable tissue, exudate level and the body massindex as covariates using PROC Mixed procedure of SAS software.

Table 16 summarizes the non-parametric analysis for mean percentreduction of wound area from Baseline to Week 4 in the ITT population.In Group A, the expected sum of score under the null hypothesis i.e. sumof scores when there is no difference in two groups was 1006.50 and theactual sum of score was 1148.00 obtained by adding the ranks of bothgroups after sorting data of wound size reduction in ascending order,and for Group B, it was 823.50 and 682.00 for expected and actual,respectively. Group A subjects had a statistically significantly higherpercent reduction of wound area than Group B subjects (p-value=0.0341).

TABLE 16 Mean Percent Reduction of Wound Area from Baseline to Week 4(Non-parametric analysis) ITT Population (N = 91) Treatment Group GroupA Group B Statistics (N = 46) (N = 45) Percent reduction of wound areafrom Baseline to Week 4 Sum of Score 1148.00 682.00 Expected Sum ofScore under Ho 1006.50 823.50 p-value ^([1]) 0.0341 Note: ^([1]) p-valuewas evaluated by using non-parametric Wilcoxon Mann-Whitney U testbetween treatment group. General Note: Mean percent reduction fromBaseline = ((baseline − week X)/baseline)*100 H_(o): Null hypothesis

The non-parametric analysis for mean percent reduction of wound areafrom Baseline to Week 4 in the PP population is summarized in Table 17The expected sum of scores, which is the sum of scores when there is nodifference in two groups, under the null hypothesis was 650.00 and theactual sum of scores, obtained by adding the ranks of both groups aftersorting data of wound size reduction in ascending order, was higher with773.00 for subjects treated with Granexin™ Gel plus SoC and in subjectstreated with SoC only, the expected sum of score was 575.00 and theactual score was lower with 452.00. The percent reduction of wound areain Group A was statistically significantly higher than Group B subjects(p-value=0.0128).

TABLE 17 Mean Percent Reduction of Wound Area from Baseline to Week 4(Non-parametric analysis) PP Population (N = 60) Treatment Group Group AGroup B Statistics (N = 32) (N = 28) Percent reduction of wound areafrom Baseline to week 4 Sum of Score 773.00 452.00 Expected under H_(o)650.00 575.00 p-value ^([1]) 0.0128 Note: ^([1]) p-value was evaluatedby using non-parametric Wilcoxon Mann-Whitney U test between treatmentgroup. General Note: Mean percent Reduction from Baseline = ((Baseline −week X)/Baseline)*100 H_(o): Null Hypothesis

The intensity of pain experienced by the subjects was recorded on aVisual Analogue Scale of 1 to 10 where 1 indicated “no pain” and 10indicated “extreme pain”. The summary of subject self assessment ofintensity of pain at all visits from Baseline to Week 12 for the ITTpopulation have been given in FIG. 2.

Table 18 summarizes the intensity of pain as assessed by the subject attreatment Week 12 of the study. There was no statistically significantdifference in the mean intensity of pain from Baseline to Week 12between the 2 treatment groups (Group A, 0.5±1.46; Group B, 0.3±0.93;p-value=0.8484).

TABLE 18 Analysis of Subject Self Assessment of Intensity of Pain atWeek 12 ITT Population (N = 91) Treatment Group Group A Group BStatistics (N = 46) (N = 45) Intensity of pain at Week 12 N 36 38 Mean0.5 0.3 SD 1.46 0.93 Median 0.0 0.0 Range (Min.:Max.) (0:6) (0:4)Missing 1 0 p-value ^([1]) 0.8484 Note: ^([1]) p-value was evaluated byusing non-parametric Wilcoxon Mann-Whitney U test between treatmentgroup.

Table 19 summarizes the intensity of pain as assessed by the subject attreatment Week 12 of the study for the PP population. There was nostatistically significant difference in the mean intensity of pain atWeek 12 between the 2 treatment groups (Group A, 0.4±1.22; Group B,0.3±0.81; p-value=0.9683). The summary of subject self assessment ofintensity of pain at all visits from Baseline to Week 12 for the PPpopulation is given in FIG. 3.

TABLE 19 Analysis of Subject Self Assessment of Intensity of Pain atWeek 12 PP Population (N = 60) Treatment Group Group A Group BStatistics (N = 32) (N = 28) Intensity of pain at Week 12 n 30 27 Mean0.4 0.3 SD 1.22 0.81 Median 0.0 0.0 Range (Min.:Max.) (0:4) (0:4)Missing 1 0 p-value ^([1]) 0.9683 Note: ^([1]) p-value was evaluated byusing non-parametric Wilcoxon Mann-Whitney U test between treatmentgroup.

The time taken for 100% wound closure for the ITT population issummarized in FIG. 4. Complete wound closure was defined as 100%epithelialization of the wound and the absence of drainage from thewound. One subject in Group A and 2 subjects in Group B had the visitdate missing and were not included in the analysis (FIG. 4). Seventeen(37.0%) subjects in Group A and 25 (55.6%) subjects in Group B werecensored. Of these, 2 (4.8%) subjects in Group B were lost-to-follow-up,1 (2.4%) subject in Group A dropped out, 9 (21.4%) subjects (Group A: 7;Group B: 2) were withdrawn, and 31 (73.8%) subjects (Group A: 10; GroupB: 21) did not have complete wound closure during the study period. Onepatient withdrew from the study but had complete wound closure and wastherefore considered in the ITT population, but excluded from the PPpopulation.

In Group A, 28 (60.9%) subjects and 18 (40.0%) subjects in Group B had100% wound closure. The median duration of 100% wound closure in Group Awas 6 weeks (90% CI was 5 to 11 weeks) and 11 weeks (90% CI: 9 to 18.29weeks) in Group B. In Group B, for 1 subject the assessments wereperformed outside the window period; the assessment was reassigned tothe closest nominal time point which was Visit 15 at 12 weeks.Statistically significantly less time was taken by subjects in Group Ato achieve 100% wound closure than Group B subjects (p-value=0.0306).

TABLE 20 Summary of Time to Complete (100%) Wound Closure by Week 12 ITTPopulation (N = 91) Treatment Group Group A Group B Statistics^([1]) (N= 46) (N = 45) Number (%) of subjects with 28 (60.9%) 18 (40.0%)complete (100%) wound closure Number (%) of censored 17 (37.0%) 25(55.6%) subjects Median duration (weeks) of 6.00 (5.00; 11.00) 11.00(9.00; 18.29) complete wound closure (90% CI)^([2]) p-value^([3]) 0.0306Note: ^([1])Percentage was calculated taking count of correspondingtreatment groups as denominator. ^([2])The median duration of completewound closure was estimated by Kaplan-Meier method and also 90%confidence interval is calculated for the median duration of 100% woundclosure. ^([3])p-value is calculated using Log-rank test for treatmentgroups using Proc Lifetest procedure of SAS software General Note: Timeto first complete wound closure where complete wound closure is definedas 100% epithelialization of the wound and the absence of drainage fromthe wound. Censoring: Subjects without occurrence of complete woundclosure during study period, drop outs and lost-to-follow up subjects iscensored at the time of last follow-up visit. The data of 1 subject inGroup A and 2 subjects in Group B were considered as missing. They hadmissing data after Screening or had missing visit date.

The time taken to complete (100%) wound closure in the PP population issummarized in Table 21 and FIG. 5 displays the Kaplan-Meier plot fortime to 100% wound closure. Of the 32 subjects in Group A, 26 (81.3%)subjects had complete (100%) wound closure and 6 (18.8%) subjects werecensored. Subjects who dropped out or were lost to follow-up werealready excluded from the PP population and subjects who did not havecomplete wound closure during the study period were censored. Theduration of wound closure was in the range of 4 weeks to 10.86 weeks andthe median duration was 6 weeks. Of the 28 subjects in Group B, 15(53.6%) subjects had complete wound closure and 13 (46.4%) subjects werecensored. The duration of wound closure was in the range of 9 weeks to18.29 weeks. The median duration of time to complete wound closure alongwith 90% CI was 14.64 weeks. The time taken for complete wound closurewas lower in Group A as compared to Group B which was statisticallysignificant (p-value=0.0057).

TABLE 21 Summary of Time to Complete (100%) Wound Closure by Week 12 PPPopulation (N = 60) Treatment Group Group A Group B Statistics^([1]) (N= 32) (N = 28) Number (%) of subjects with 26 (81.3%) 15 (53.6%)complete (100%) wound closure Number (%) of censored 6 (18.8%) 13(46.4%) subjects Median duration (weeks) of 6.00 (4.00; 10.86) 14.64(9.00; 18.29) complete wound closure (90% CI) ^([2]) p-value^([3])0.0057 Note: ^([1])Percentage was calculated taking count ofcorresponding treatment groups as denominator ^([2]) The median durationof complete wound closure was estimated by Kaplan-Meier method and also90% confidence interval was calculated for the median duration of 100%wound closure. ^([3])p-value was calculated using Log-rank test fortreatment groups using Proc Lifetestt procedure of SAS software

The time taken to complete 50% wound closure by Week 12 in the ITTpopulation is summarized in Table 22 and FIG. 6 displays theKaplan-Meier plot for time to 50% wound closure in the ITT population.One subject in Group A and 2 subjects in Group B had the visit datemissing and were not included in the analysis (FIG. 6). In Group A, 9(19.6%) subjects and 11 (24.4%) subjects in Group B were censored.Overall, of the 20 censored subjects, 2 (10.0%) subjects werelost-to-follow-up, 1 (5.0%) subject dropped out, 9 (45.0%) subjects werewithdrawn, and 8 (40.0%) subjects did not have 50% wound closure duringthe study period.

Thirty six (78.3%) subjects in Group A and 32 (71.1%) subjects in GroupB had 50% wound closure. The median duration of 50% wound closure was 2weeks (90% CI was 2 to 3.14 weeks) in Group A and 3.29 weeks (90% CI was3 to 4.14 weeks) in Group B. The time taken for 50% wound closure wasnot statistically significantly lower in Group A than Group B(p-value=0.5433).

TABLE 22 Summary of Time to Complete 50% Wound Closure by Week 12 ITTPopulation (N = 91) Treatment Group Group A Group B Statistics ^([1]) (N= 46) (N = 45) Number (%) of subjects with 36 (78.3%) 32 (71.1%) 50%wound closure Number (%) of censored 9 (19.6%) 11 (24.4%) subjectsMedian duration (weeks) of 2.00 (2.00; 3.14) 3.29 (3.00; 4.14) completewound closure (90% CI) ^([2]) p-value ^([3]) 0.5433 Note: ^([1])Percentage was calculated taking count of corresponding treatment groupsas denominator ^([2]) The median duration of complete wound closure wasestimated by Kaplan-Meier method and also 90% confidence interval iscalculated for the median duration of 50% wound closure. ^([3]) p-valuewas calculated using Log-rank test for treatment groups using ProcLifetestt procedure of SAS software

The time taken to complete 50% wound closure in the PP population issummarized in Table 23 and FIG. 7 displays the Kaplan-Meier plot fortime to 50% wound closure in the PP population. Of the 32 subjects inGroup A, 30 (93.8%) subjects completed 50% wound closure and 2 (6.3%)subjects were censored. In Group A, the duration of wound closure was inthe range of 2 weeks to 3.14 weeks and the median duration was 2 weeks.Of the 28 subjects in Group B, 25 (89.3%) subjects had 50% wound closureand 3 (10.7%) subjects who did not have at least 50% wound closureduring the study period were censored. The duration of wound closure wasin the range of 3 weeks to 5 weeks. The median duration of time tocomplete 50% wound closure was 4.07 weeks. The time taken for completewound closure was not statistically significantly lower in subjects inGroup A as compared to the time taken by subjects in Group B(p-value=0.2084).

TABLE 23 Summary of Time to Complete (50%) Wound Closure by Week 12 PPPopulation (N = 60) Treatment Group Group A Group B Statistics^([1]) (N= 32) (N = 28) Number (%) of subjects with 30 (93.8%) 25 (89.3%) 50%wound closure Number (%) of censored 2 (6.3%) 3 (10.7%) subjects Medianduration (weeks) of 2.00 (2.00; 3.14) 4.07 (3.00; 5.00) complete woundclosure (90% CI)^([2]) p-value^([3]) 0.2084 Note: ^([1])Percentage wascalculated taking count of corresponding treatment groups as denominator^([2])The median duration of complete wound closure is estimated byKaplan-Meier method and also 90% confidence interval is calculated forthe median duration of 100% wound closure. ^([3])p-value is calculatedusing Log-rank test for treatment groups using Proc Lifetestt procedureof SAS software

Additional to the Kaplan-Meier Model, Cox Proportional Hazard RegressionModel was used to compare the event-time distribution function betweenthe two treatment groups. This analysis was conducted to evaluate if oneor more covariates were associated with the time to complete 100% or 50%wound closure. Table 24 summarizes the Cox Proportional HazardRegression Analysis of time to complete wound closure in the ITTpopulation. The effect of covariates estimated by the proportionalhazard model is reported as hazard ratios. Though treatment was not asignificant factor affecting wound closure (p-value=0.2903), thesubjects treated with Granexin™ Gel plus SoC would have wound closure1.568 times more than the subjects treated with SoC only. All otherparameters, including wound size, wound duration, baseline wound depth,and use of dressings did not affect wound closure in both the treatmentgroups.

TABLE 24 Analysis of Time to Complete (100%) Wound Closure ITTPopulation (N = 91) Hazard 95% CI for Hazard Ratio Statistics^([1])Ratio^([1]) Lower Upper p-value Treatment group 1.568 0.681 3.607 0.2903Wound size 0.999 0.997 1.000 0.1265 Wound duration 0.995 0.976 1.0130.5754 Baseline wound depth 0.782 0.239 2.558 0.6839 BMI 0.966 0.8611.084 0.5592 Use of dressings — — — — Note: ^([1])Cox ProportionalHazard ratio with 95% confidence interval and p-value was calculatedusing PROC PHREG procedure.

Cox Proportional Hazard Regression Analysis for time to 100% woundclosure in the PP population (Table 25) shows that though treatment wasnot a significant factor affecting wound closure (p-value=0.0856), thesubjects treated with Granexin™ Gel plus SoC would have wound closure2.339 times more than the subjects treated with SoC only. Similarly,baseline wound depth and BMI were not significant factors (baselinewound depth: p-value=0.4554, BMI: p-value=0.4435) but would affect woundclosure by 1.715 times and 1.050 times, respectively. All otherparameters, including wound size, wound duration, and use of dressingsdid not affect wound closure in both the treatment groups.

TABLE 25 Analysis of Time to Complete (100%) Wound Closure (Cox'sProportional Hazards Regression Analysis) PP Population (N = 60) 95% CIfor Hazard Hazard Ratio Statistics^([1]) Ratio^([1]) Lower Upper p-valueTreatment group 2.339 0.888 6.161 0.0856 Wound size 0.999 0.997 1.0000.1703 Wound duration 0.992 0.974 1.011 0.4177 Baseline wound depth1.715 0.416 7.071 0.4554 BMI 1.050 0.927 1.190 0.4435 Use of dressings —— — — Note: ^([1])Cox Proportional Hazard ratio with 95% confidenceinterval and p-value was calculated using PROC PHREG procedure.

None of the covariates such as treatment group, wound size, woundduration, baseline wound depth, and BMI were significantly associatedwith time to 50% wound closure. However, in the PP population, subjectstreated with Granexin™ Gel plus SoC would have wound closure 1.186 timesmore than subjects in Group B.

FIG. 8 and FIG. 9 display the average wound closure in the ITT and PPpopulations in the study, respectively.

Safety Evaluation: Brief Summary of Adverse Events

Of 92 subjects, 71 (77.2%) subjects did not report any AEs and 21(22.8%) subjects reported 26 AEs. Out of 26 AEs, 18 (69.2%) were mildand 2 (15.4%) events in each treatment group were moderate and severe,respectively. A total of 21 (80.8%) AEs were not related and 5 (19.2%)AEs were unlikely related to the study drug or treatment. None of theAEs were related to the study drug. Of the 26 events, 18 (69.2%)recovered and 3 (11.5%) events recovered with sequelae. No action wastaken against 22 AEs. None of the subjects withdrew due to an AE.

A total of 5 subjects, of which 2 (4.3%) subjects were in Group A and 3(6.5%) subjects in Group B, reported 5 SAEs. One subject in Group A dieddue to an unknown cause and 1 subject in Group B died due to myocardialinfarction (Table 26).

TABLE 26 Overview of Adverse Events-Safety Population (N = 92) TreatmentGroup Group A Group B Overall Statistics (N = 46) (N = 46) (N = 92)Total number of AEs reported 13 13 26 Subjects reporting Any 11 (23.9%)10 (21.7%) 21 (22.8%) AEs^([1]) Subjects reporting 1 AE  9 (19.6%)  7(15.2) 16 (17.4%) Subjects reporting >1 AE  2 (4.3%)  3 (6.5%)  5 (5.4%)Subjects reporting No AEs^([1]) 35 (76.1%) 36 (78.3%) 71 (77.2%) Numberof AEs with severity of:^([2]) Mild 11 9 (69.2%)  9 (69.2%) 18 (69.2%)Moderate  2 (15.4%)  2 (15.4%)  4 (15.4%) Severe  2 (15.4%))  2 (15.4%) 4 (15.4%) Number of AEs with relationship of^(:[2]) Not Related 10(76.9%) 11 (84.6%) 21 (80.8%) Unlikely  3 (23.1%)  2 (15.4%)  5 (19.2%)Possible  0 (0.0%)  0 (0.0%)  0 (0.0%) Probable  0 (0.0%)  0 (0.0%)  0(0.0%) Definite  0 (0.0%)  0 (0.0%)  0 (0.0%) Number of AEs byoutcome:[^(2]) Recovered  7 (53.8%) 11 (84.6%) 18 (69.2%) Recovered withsequelae  2 (15.4%)  1 (7.7%)  3 (11.5%) Ongoing when subject  3 (23.1%) 0 (0.0%)  3 (11.5%) completed the study Death  1 (7.7%)  1 (7.7%)  2(7.7%) Unknown  0  0  0 Number of AEs by action taken:^([2]) None 11(84.6%) 11 (84.6%) 22 (84.6%) Discontinued study drug  2 (15.4%)  2(15.4%)  4 (15.4%) Subjects reporting AEs  0  0  0 leading towithdrawal^([1]) Subjects Reporting SAEs^([1])  2 (4.3%)  3 (6.5%)  5(5.4%) Subject reporting death  1 (2.2%)  1 (2.2%)  2 (2.2%) Note:^([1])Percentage was calculated by taking respective column header groupcount as denominator. ^([2])Percentage was calculated by taking count of‘Total Number of AEs Reported’ in corresponding treatment group asdenominator.

The AEs by MedDRA system organ class and preferred term are summarizedin Table 27. There was no statistically significant difference in thenumber of subjects with at least 1 AE between the 2 treatment groups(p-value=0.8038). In Group A, more than 5% study population reportedwound complication (Group A: 5 [10.9%] subjects) and headache (Group A:4 [8.7%] subjects) as AEs in this study.

TABLE 27 Summary of Adverse Events by MedDRA System Organ Class andPreferred Term Safety Population (N = 92) Treatment Group, n (%)^([1])Group A Group B Overall System Organ Class/Preferred Term (N = 46) (N =46) (N = 92) p-value^([2]) Total number of subjects with at 11 (23.9%)10 (21.7%) 21 (22.8%) 0.8038 least one AEs Total number of AEs reported13 13 26 Cardiac disorders  0  1 (2.2%)  1 (1.1%) Myocardialinfarction^([3])  0 [0]  1 (2.2%) [1]  1 (1.1%) [1] Gastrointestinaldisorders  0  1 (2.2%)  1 (1.1%) Toothache  0 [0]  1 (2.2%) [1]  1(1.1%) [1] General disorders and  1 (2.2%)  0  1 (1.1%) administrationsite conditions Death  1 (2.2%) [1]  0 [0]  1 (1.1%) [1] Infections andinfestations  0  2 (4.3%)  2 (2.2%) Cellulitis  0 [0]  1 (2.2%) [1]  1(1.1%) [1] Respiratory tract infection  0 [0]  1 (2.2%) [1]  1 (1.1%)[1] Injury, poisoning and procedural  6 (13.0%)  2 (4.3%)  8 (8.7%)complications Wound complication  5 (10.9%) [6]  2 (4.3%) [3]  7 (7.6%)[9] Foot fracture  1 (2.2%) [1]  0 [0]  1 (1.1%) [1] Metabolism andnutrition disorders  0  1 (2.2%)  1 (1.1) Diabetes mellitus inadequate 0 [0]  1 (2.2%) [1]  1 (1.1%) [1] control Musculoskeletal andconnective  0 (6.5%)  3 (3.3%) tissue disorders Back pain  0 [0]  1(2.2%) [1]  1 (1.1%) [1] Pain in extremity  0 [0]  2 (4.3%) [2]  2(2.2%) [2] Nervous system disorders  4 (8.7%)  1 (2.2%)  5 (5.4%)Headache  4 (8.7%) [4]  1 (2.2%) [1]  5 (5.4%) [5] Respiratory, thoracicand  1 (2.2%)  0  1 (1.1%) mediastinal disorders Pneumonitis  1 (2.2%)[1]  0 [0]  1 (1.1%) [1] Skin and subcutaneous tissue  0  1 (2.2)  1(1.1%) disorders Blister  0 [0]  1 (2.2%) [1]  1 (1.1%) [1] Note:^([1])Percentage was calculated by taking respective column header groupcount as denominator. ^([2])p-value was calculated by comparing twotreatment group using Chi-square test. ^([3])Myocardial infarction ledto death of the subject

Out of 26 AEs, 2 events each in both the treatment groups wereconsidered severe AEs. In both Group A and B, 9 (69.2%) AEs each weremild, 2 (15.4%) AEs each were moderate and 2 (15.4%) AEs each weresevere. The AEs with severe intensity were death and foot fracture inGroup A and myocardial infarction and inadequate control of diabetesmellitus in Group B. None of the severe AEs were related to the studydrug or study treatment

Overall, 10 (76.9%) AEs in Group A and 11 (84.6%) AEs in Group B werenot related, 3 (23.1%) AEs in Group A and 2 (15.4%) AEs in Group B wereunlikely related. None of the AEs were related to Granexin™ Gel or studytreatment.

Two AEs of death (Subject 903) and foot fracture (Subject 504) in GroupA led to study drug discontinuation. In Group B, two events ofcellulitis (Subject 524) and wound complication (Subject 806 withincrease in size of ulcer) led to discontinuation of study treatment.

Three events recovered with sequelae (Group A, foot fracture [Subject504] and wound complication [Subject 508]; Group B, right footcellulitis [Subject 524]). One subject in each treatment group reporteddeath (Group A, unknown cause [Subject 903]; Group B, myocardialinfarction [Subject 827]).

Table 28 summarizes SAEs by MedDRA system organ class and preferredterm. Five (5.4%) subjects (Group A: 2 subjects; Group B: 3 subjects)reported SAEs where foot fracture (Subject 504) and death due to unknowncause (Subject 903) were reported by Group A subjects; and myocardialinfarction (Subject 827), diabetes mellitus inadequate control (Subject201), and cellulitis (Subject 524) were reported by Group B subjects.

TABLE 28 Summary of Serious Adverse Events by MedDRA System Organ Classand Preferred Term Safety Population (N = 92) Treatment Group, n(%)^([1]) Group A Group B Overall System Organ Class/Preferred Term (N =46) (N = 46) (N = 92) Total number of subjects with 2 (4.3) 3 (6.5) 5(5.4) at least one SAE Total number of SAEs 2 3 5 Cardiac disorders 0(0.0) 1 (2.2) 1 (1.1) Myocardial infarction^([2]) 0 (0.0) [0] 1 (2.2)[1] 1 (1.1) [1] General disorders and administration 1 (2.2) 0 (0.0) 1(1.1) site conditions Death 1 (2.2) [1] 0 (0.0) [0] 1 (1.1) [1]Infections and infestations 0 (0.0) 1 (2.2) 1 (1.1) Cellulitis 0 (0.0)[0] 1 (2.2) [1] 1 (1.1) [1] Injury, poisoning and procedural 1 (2.2) 0(0.0) 1 (1.1) complications Foot fracture 1 (2.2) [1] 0 (0.0) [0] 1(1.1) [1] Metabolism and nutrition disorders 0 (0.0) 1 (2.2) 1 (1.1)Diabetes mellitus inadequate control 0 (0.0) [0] 1 (2.2) [1] 1 (1.1) [1]Note: ^([1])Percentage was calculated by taking respective column headergroup count as denominator. ^([2])Myocardial infarction led to death ofthe subject.

The 5 SAEs with the outcome are summarized in Table 29 below:

TABLE 29 SAEs by Severity, Outcome, and Action Taken Relationship toSubject Study Drug/ ID Severity Study Treatment Action Taken Outcome 903Severe Not related None Fatal 827 Severe Not related None Fatal 201Severe Not related None Recovered 504 Severe Not related DiscontinuedRecovered with Study drug sequelae 524 Moderate Not related DiscontinuedRecovered with Study drug sequelae

Safety Conclusions: Overall, 21 subjects reported 26 AEs of whichmajority were mild in intensity. A total of 4 moderate and 4 severe AEswere reported in this study. In Group A, death and foot fracture werethe severe AEs and myocardial infarction and inadequate control ofdiabetes mellitus were severe AEs reported in Group B. All the severeAEs were not related to study treatment. None of the AEs reported wererelated to Granexin™ Gel or study treatment. Of the 26 events, 18(69.2%) recovered and 3 (11.5%) events recovered with sequelae. Noaction was taken against 22 AEs. Study drug or treatment wasdiscontinued in a 2 subjects each in Group A and Group B. None of thesubjects withdrew due to an AE. Five subjects reported 5 SAEs. None ofthe SAEs, including 1 fatal event in Group A, were related to Granexin™Gel.

Discussion and Overall Conclusions:

This was a double blind, randomized, prospective, parallel group,multi-center Phase II study conducted at 8 centers in India to evaluatethe efficacy and safety of Granexin™ Gel in the treatment of DFU. Thetotal duration of the study was 12 months, including 6 months enrollmentperiod and 6 months for study procedures (3 months each for efficacy andsafety analyses). Hence, the study duration for an individual subjectwas approximately 6 months, including 18 visits. For interim analysis,data up to 12 weeks (Visit 15) was considered. A total of 98 subjectswith DFU were screened for this study and 6 were found to be screenfailures. The remaining 92 subjects were randomized in the study toreceive Granexin™ Gel plus SoC or only SoC in the treatment of DFU. Ofthese subjects, 76 (82.6%) completed the study treatment visits up toVisit 15 and are now in the follow-up stage of the study.

The primary efficacy endpoint of the study was to evaluate mean percentwound closure from Baseline to Week 12. The non-parametric WilcoxonMann-Whitney U test was used to analyze mean percent reduction of woundarea from Baseline to Week 12 in both the treatment groups since thedata did not follow a normal distribution, as confirmed by aShapiro-Wilk p-value <0.0001 and a non-linear Q-Q plot. In the PPpopulation, subjects treated with Granexin™ Gel plus SoC (Group A)showed statistically significantly higher percent wound closure fromBaseline to Week 12 compared to subjects treated with SoC alone (GroupB) (p value=0.0069), suggesting that at Week 12 wounds treated withGranexin™ Gel plus SoC healed better than the wounds treated with SoCalone.

The secondary endpoints of the study were to assess mean percent woundclosure at Week 4, subject assessment of pain, and time to completewound closure. At Week 4 in the PP population, Group A subjects showedstatistically significantly higher percent reduction of wound area fromBaseline compared to Group B subjects (p-value=0.0128; WilcoxonMann-Whitney U test). There was no statistically significant differencein intensity of pain as assessed by the subject from Baseline to Week 12between the 2 treatment groups (p-value=0.8484; Wilcoxon Mann-Whitney Utest), hence Granexin™ Gel did not affect the intensity of painexperienced by the subjects as recorded on a Visual Analogue Scale of 1to 10.

Complete wound closure was defined as 100% epithelialization of thewound and the absence of drainage from the wound. Group A subjects inthe PP population took significantly less time to achieve 100% woundclosure than Group B subjects in the PP population (median duration:Group A, 6 weeks; Group B, 14.64 weeks; p-value=0.0057; log-rank test).However, in the PP population the time taken for 50% wound closure wasnot significantly lower in Group A as compared to Group B (medianduration: Group A, 2 weeks; Group B, 4.07 weeks; p-value=0.2084;log-rank test). The categorical analysis of incidence of 100% and 50%wound closure at Week 12 was conducted and the subjects who had 100%wound closure were considered as responders. The number of responders inGroup A in the PP population was significantly higher than in Group B(100% wound closure: p-value=0.0104; 50% wound closure: p-value=0.0063;Chi-square analysis), indicating that the wounds treated with Granexin™Gel plus SoC have a higher frequency of 100% and 50% epithelializationthan wounds treated with SoC alone.

Cox Proportional Hazard Regression Model was used to compare theevent-time distribution function between the treatment groups. Thisanalysis was conducted to evaluate if one or more covariates wereassociated with the time to complete 100% or 50% wound closure. CoxProportional Hazard Regression Analysis for time to 100% wound closureshowed that in the PP population though treatment was not a significantfactor affecting wound closure. Group A subjects demonstrated 2.3 timeshigher wound closure than Group B subjects. In addition, baseline wounddepth and BMI were not significant factors but affected wound closure by1.715 times and 1.050 times, respectively. All other parameters,including wound size, wound duration, and use of dressings did not havea statistically significant difference in affecting wound closure forboth the treatments. Cox Proportional Hazard Regression Analysis fortime to 50% wound closure showed that none of the covariates (treatmentgroup, wound size, wound duration, baseline wound depth, and BMI) weresignificantly associated with time to 50% wound closure. However, GroupA subjects had 1.186 times more wound closure than Group B subjects.

The safety variable was to evaluate the incidence of treatment relatedAEs. Out of 92 subjects, 21 (22.8%) subjects reported 26 AEs. There wasno significant difference in the number of subjects with at least 1 AEbetween the 2 treatment groups (Group A, 11; Group B, 10;p-value=0.8038). A total of 4 AEs were of severe intensity. None of theAEs were related to the Granexin™ Gel. None of the subjects withdrew dueto an AE. A total of 5 (5.4%) subjects reported 5 SAEs of which 2 wereGroup A subjects and 3 were Group B subjects, none of the SAEs wasrelated to Granexin™ Gel or study treatment. In Group A, 1 death wasreported due to an unknown cause and 1 death in Group B occurred due tomyocardial infarction.

In conclusion, Granexin™ Gel along with SoC has shown to acceleratewound healing in subjects with DFU, making it an efficacious, safe, andwell tolerated therapeutic option for the treatment of DFU.

Example 4 Treatment of Venous Leg Ulcers

A Phase II, Randomized, Prospective, Double Blind, Parallel Group,Multi-Center Study to Determine the Safety and Efficacy of Granexin™ Gelin the Treatment of Venous Leg Ul

This was a double blind, multi-center, phase II study to evaluate theefficacy and safety of Granexin™ Gel in the treatment of Venous LegUlcer (VLU). Total duration of the study was 11 months. The enrollmentperiod of the study was 5 months and 6 months for study procedures.Study duration for individual subjects was 6 months and each subject wasto attend a total of 18 visits during the study. Subjects wererandomized to 1 of 2 treatment groups: Group A: Granexin™ Gel plusStandard Of Care (SoC) in treatment of VLU Group B: SoC in treatment ofVLU Subjects in Group A received Granexin™ Gel (100 μM ACT1 peptide)plus SoC compared to SoC alone in Group B. The total study duration forefficacy assessments was 12 weeks with additional 12 weeks follow up forsafety evaluations. The study procedures were divided into 3 phases:Screening Phase (Day −7 through Day 0), Treatment Phase (Day 0 throughWeek 12), and Follow-up Phase (Month 4 through Month 6).

Subjects enrolled in the study may have had multiple ulcers but only oneulcer was considered as the target ulcer. According to the randomizationschedule, subjects had their ulcer site treated with either Granexin™Gel and SoC or SoC alone. Each subject in Group A was treated up to 14applications of Granexin™ Gel over the initial 12 weeks of the studyperiod. Other than the application of Granexin™ Gel for Group Asubjects, management of the wound sites was similar in both groups.

Each subject was assessed weekly for the first 12 weeks, followed bymonthly assessments for the next 3 months. Wound photographs were usedto assess wound closure in all subjects. Safety was determined byTreatment Emergent Adverse Events (TEAEs).

The primary end point of the study is:

The mean percent wound closure from Baseline to 12 weeks

The secondary end points are:

Mean percent wound closure at 4 weeks

Time to 50% wound closure

Time to complete wound closure where complete wound closure will bedefined as 100% epithelialization of the wound and the absence ofdrainage from the wound

Subject self-assessment of intensity of pain

Incidence of treatment related adverse events (AE)

Inclusion Criteria:

A subject was enrolled in this study if he/she met the followingcriteria:

-   2. Male or female aged 18 years and older-   2a. Female subjects were post-menopausal or surgically sterilized,    or-   2b. Female of child-bearing potential must have had a negative    pregnancy test at Screening, and agreed to use hormonal    contraceptive or intra-uterine device or diaphragm with spermicide    or condom with spermicide or abstinence throughout the study-   3. Subjects with ulcers of venous origin, as clinically determined    by the Investigator by a positive venous reflux test (venous    refilling <20 seconds) using Doppler Ultrasound for at least 4 weeks    prior to screening day, which have not adequately responded to    conventional ulcer therapy-   4. Ulcers that extend through the epidermis but not through muscle,    tendon, or bone (Stage II or III ulcers as defined by the    International Association of Enterostomal Therapists)-   5. Surface ulcer between 0.5 cm² and 40 cm² post debridement-   6. Signed informed consent form

Exclusion Criteria:

-   A subject was not eligible for this study if he/she met any of the    following criteria:-   1. Decrease or increase in the ulcer size by 30% or more during 7    day screening period-   2. Cannot tolerate or comply with compression therapy-   3. An ulcer which showed signs of severe clinical infection, defined    as pus oozing from ulcer site-   4. The ulcer to be treated required operative debridement-   5. An ulcer positive for β-hemolytic Streptococcus upon culture-   6. The ulcer had more than 50% slough, significant necrotic tissue,    osteomyelitis, bone, tendon, or capsule exposure or avascular ulcer    beds-   7. Highly exuding wounds (wounds that required a daily dressing    change) df-   8. Ankle Brachial Pressure Index (ABPI)<0.65-   9. With active systemic infections-   10. With clinically significant medical conditions as determined by    the Investigator which would impair wound healing including renal,    hepatic, hematologic, neurologic, or immune disease. Examples    included but were not limited to:    a. Renal insufficiency as an estimated glomerular filtration rate    which was <30 mL/min/1.7 m²    b. Abnormal blood biochemistry defined as 3 times that of the upper    limit of the normal range    c. Hepatic insufficiency defined as total bilirubin >2 mg/dL or    serum albumin <25 g/L    d. HbA1c>9.0%    e. Hemoglobin <10 g/dL    f. Hematocrit <0.30    g. Platelet count <100,000-   11. Presence of an active systemic or local cancer or tumor of any    kind (with the exception of non-melanoma skin cancer)-   12. With active osteomyelitis of the study foot-   13. With severe rheumatoid arthritis (with more than 20 persistently    inflamed joints, or below lower normal limit blood albumin level, or    evidence of bone and cartilage damage on X-ray, or inflammation in    tissues other than joints) and other collagen vascular diseases-   14. With active connective tissue disease-   15. Treatment with systemic corticosteroids (>15 mg/day), or current    immunosuppressive agents-   16. Previous or current radiation therapy or likelihood to receive    this therapy during study participation-   17. Pregnant or nursing subjects-   18. Known prior inability or unavailability to complete required    study visits during study participation-   19. A psychiatric condition (e.g. suicidal ideation) or chronic    alcohol or drug abuse problem, determined from the subject's medical    history, which, in the opinion of the Investigator, may pose a    threat to subject compliance-   20. Use of any investigational drug or therapy within the 28 days    prior to Screening-   21. Any other factor which may have, in the opinion of the    Investigator, compromised participation and follow-up in this study

Granexin™ Gel (100 μM ACT1 peptide) was applied topically on the woundon Day 0 (Baseline visit), Day 3, Week 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, and 12. The duration of the study was 11 months which included 5months enrollment period. Individual subject participation wasapproximately for the duration of 6 months.

The reference therapy used in this study was SoC for VLU. The ulcer wascleaned with sterile saline and bleeding was stopped before dressing.The ulcer was dressed according to the following regimen:

Applied a non-adherent dressing over the ulcer which extended 0.5 inchbeyond the ulcer perimeter and inflamed skin marginsApplied a non-occlusive dressing such as fine mesh gauze which waseither folded or rolled as a bolsterApplied a self adherent high compression four layer elastic wrap (3MElastic Adhesive Bandage) or a compression crepe bandage frommetatarsals to tibial plateau so that therapeutic compression is appliedto the ulcer site

This was a double blind, randomized, prospective, parallel group,multi-center Phase II study conducted at 10 centers in India to evaluatethe efficacy and safety of Granexin™ Gel in the treatment of VLU. Thetotal duration of the study was 11 months, including 5 months enrollmentperiod and 6 months for study procedures. The study duration forindividual subjects was 6 months which included 18 visits. A total of101 subjects with VLU were screened for this study, 5 were found to bescreen failures, and 4 subjects did not initiate participation in thestudy. A total of 92 subjects enrolled in the study had mean woundduration of 68.4 weeks at Screening and mean wound area of 353.64 mm² atBaseline. These subjects were randomized to receive Granexin™ Gel plusSoC or SoC alone for the treatment of VLU. Of these, 70 completed thestudy and 22 subjects did not complete the study due to not meetingexclusion criterion number 1, consent withdrawal, loss to follow-up,non-compliance, or SAE. Of 92 enrolled subjects, 15 subjects metexclusion criterion 1 but were considered in the Safety and ITTpopulation. These subjects were excluded from the mITT and PPpopulations. A total of 83 (90.2%) subjects were males and 9 (9.8%) werefemales. The mean age of the study population was 49.8 years, meanweight was 70.47 kg, mean height was 168.35 cms, and mean BMI was 24.79kg/m².

This was a phase II, prospective, randomized, double blind, parallelgroup, and multi-center study conducted in India in subjects with VLU.The total study duration was 11 months. The enrollment period was 5months and 6 months for study procedures. Study duration for individualsubjects was 6 months and each subject attended a total of 18 visits,including Screening (Visit 1), Baseline, (Visit 2), Treatment (Visit3-15), and Follow-up (Visit 16-18) visits. The study procedures weredivided into 3 phases: Screening Phase (Days −7 through Day 0),Treatment Phase (Day 0 through Week 12), and Follow-up Phase (Months 4through Month 6). Subjects were randomized to 1 of the 2 treatmentgroups:

Group A: Granexin™ Gel plus SoC

Group B: SoC only

Only one ulcer per subject was considered as the target ulcer which wasthe largest ulcer. Each subject in the Granexin™ Gel treatment group wastreated with up to 14 applications of Granexin™ Gel over the initial 12weeks of the study period. Other than the application of Granexin™ Gel,management of ulcer was similar in both the groups.

The primary efficacy endpoint of the study was to evaluate mean percentwound closure from Baseline (Visit 2) to Week 12 (Visit 15). Thenon-parametric Wilcoxon Mann-Whitney U test was used to analyze meanpercent reduction of wound area from Baseline to Week 12 in both thetreatment groups since the data did not follow a normal distribution, asconfirmed by a Shapiro-Wilk p-value<0.0001 and a non-linear Q-Q plot.Subjects treated with Granexin™ Gel plus SoC showed statisticallysignificantly higher percent wound closure from Baseline to Week 12compared to subjects treated with SoC alone (ITT p-value=0.0238; mITTp-value=0.0105; PP p-value=0.0073), suggesting that at Week 12, woundstreated with Granexin™ Gel plus SoC reduced in size when compared towounds treated with SoC alone.

Mean percent wound closure at Week 4, time to complete (100% and 50%)wound closure, incidence of complete wound closure, and subject selfassessment of pain were also analyzed. At Week 4, a higher percentagereduction of wound area was observed in Granexin™ Gel plus SoC groupthan in the SoC group and this was statistically significant (ITTp-value=0.0006; mITT p-value=0.0001; PP p-value<0.0001 WilcoxonMann-Whitney U test).

Subjects treated with Granexin™ Gel plus SoC achieved 100% wound closurein median duration of 6 weeks in ITT, mITT, and PP population which wassignificantly faster compared to the SoC group in which the medianduration was 12.14 weeks in the ITT population and not achieved in bothmITT and PP population during the 12 weeks of efficacy assessments (ITTp-value=0.0006; mITT p-value=0.0001; PP p-value<0.0001; log-rank test).

Subjects treated with Granexin™ Gel plus SoC achieved 50% wound closurein 2.86 weeks in ITT, mITT and PP population while subjects treated withSoC alone achieved it in 6.86 weeks in ITT population and 8 weeks inboth mITT and PP population (ITT p-value=0.0002, mITT p-value<0.0001, PPp-value<0.0001). The categorical analysis of incidence of 100% and 50%wound closure at Week 12 was conducted and the subjects who had 100% and50% wound closure were considered as responders. Overall in the ITT,mITT, and PP populations, 56.5%, 63.4%, and 74.3% of the subjects inGranexin™ Gel plus SoC group, respectively, responded to treatment with100% wound closure as compared to 30.3% of the subjects in the SoCgroup. The number of responders were significantly higher in Granexin™Gel plus SoC group than in the SoC group (ITT: p-value=0.0061; mITTp-value=0.0040; PP: p-value=0.0003; Chi-square analysis). Similarly, inthe PP population, 80.0% of the subjects in Granexin™ Gel plus SoC groupresponded to treatment and achieved 50% wound closure as compared to51.5% of the subjects in the SoC group. The number of responders with50% wound closure were significantly higher in Granexin™ Gel plus SoCgroup than in the SoC group (p-value=0.0131; Chi-square analysis),indicating that the wounds treated with Granexin™ Gel plus SoC havehigher incidences of 100% and 50% wound closure (epithelialization) thanwounds treated with SoC alone.

Cox Proportional Hazard Regression Model analysis was conducted toevaluate if one or more covariates such as treatment group, woundduration, baseline wound depth, and BMI etc. were associated with thetime to complete 100% or 50% wound closure. It showed that treatmentgroup was a significant factor affecting wound closure and Granexin™ Gelplus SoC group subjects had ITT: 2.281; mITT: 2.776; PP: 3.150 timesmore chances of 100% wound closure and ITT: 2.247; mITT: 2.501; PP: 2.61times more chances of 50% wound closure than the SoC group subjects.

At every visit, self assessment of intensity of pain score was completedby each subject and the scores were given in comparison to the lastvisit. Therefore significant change in the pain scores was not observedand there was no statistically significant difference in intensity ofpain as assessed by the subject at Week 12 between the 2 treatmentgroups.

The findings from the present study demonstrated a good safety profilewith Granexin™ Gel. Overall, 28 subjects reported 40 AEs of which 24 AEswere reported by subjects in Granexin™ Gel plus SoC group; 16 AEs werereported in the SOC group. The only 2 SAEs reported, were in the SoCgroup. One SAE was infarction which resulted in death, and the other SAEwas Deep Vein Thrombosis. Of the 40 AEs, 35 (87.5%) recovered. None ofthe AEs were related to Granexin™ Gel. None of the subjects werewithdrawn from the study due to an AE.

No clinically significant abnormalities in the laboratory parameterswere observed in either of the treatment groups. The vital signs and ECGwere normal and comparable between both the treatment groups. Anti-ACT1peptide antibodies were not detected in the serum samples collected atScreening or at Week 12.

In conclusion, Granexin™ Gel along with SoC has shown to acceleratewound healing in subjects with VLU, making it an efficacious, safe, andwell tolerated therapeutic option in the treatment of VLU.

Description of Study Visits: The description of 18 visits along withpremature study discontinuation and unscheduled visits is summarizedbelow:

Visit 1: Screening Visit (Day −7): The Screening visit was held amaximum of 7 days prior to the Baseline visit.

Visit 2: Baseline Visit (Day 0)

The Baseline visit was conducted within 7 days of the Screening visit.If wound size increased or decreased by 30% on the day of randomizationfrom Screening (exclusion criterion number 1), then the subject wasconsidered ineligible for participation in the study. The followingprocedures were performed at the Baseline visit:

Visit 3-14: Treatment Visits (Day 3 Through Week 11)

The treatment visits were conducted at Day 3 and at Week 1, 2, 3, 4, 5,6, 7, 8, 9, 10, and 11 after Baseline visit ±1 day. The followingprocedures were performed at these treatment visits:

Visit 15: End of Treatment Visit (Week 12)

Visit 16-18: Follow-up Visits including End of Study Visit (Month 4through 6):

The Follow-up visits were conducted at Month 4±3 days, Month 5±3 days,and Month 6±3 days (End of Study [EOS] visit). The following procedureswere performed at this visit:

Standard of Care Treatment

The SoC was followed in both treatment groups. The ulcer was cleanedwith sterile saline and bleeding was stopped before dressing. The ulcerwas dressed according to the following regimen:

Applied a non-adherent dressing over the ulcer which extended 0.5 inchbeyond the ulcer perimeter and inflamed skin marginsApplied a non-occlusive dressing such as fine mesh gauze which waseither folded or rolled as a bolsterApplied a self adherent high compression four layer elastic wrap (3MElastic Adhesive Bandage) or a compression crepe bandage frommetatarsals to tibial plateau so that therapeutic compression is appliedto the ulcer site

In the Granexin™ Gel plus SoC group, the SoC as mentioned above wasfollowed along with the application of Granexin™ Gel. The ulcer wascleaned with sterile saline and bleeding was stopped before applying theGranexin™ Gel on Days 0 and 3, and Weeks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, and 12. The Granexin™ Gel was dispensed directly from its 5 ml glassvial(s) to completely cover the wound bed. With a clean sterile cottonswab, sterile tongue depressor, or a similar application aid, theGranexin™ Gel was evenly spread over the wound surface.

Granexin™ Gel is a topical gel with the active drug substanceAlpha-Connexin C-terminal peptide 1 (ACT1) peptide developed atFirstString Research for the management of wounds. ACT1 is a syntheticpeptide (25 amino acid) designed to mimic the Carboxy (C)-terminus ofthe ubiquitous transmembrane gap junction protein Connexin 43 (Cx43),with high binding specificity to zona occludens, a cytoplasmic tightjunction protein. While the mechanism of action of ACT1 has not beencompletely elucidated, the peptide is soluble and engineered to directlytranslocate within cells. ACT1 peptide interacts with a known bindingpartner of Cx43—the Postsynaptic Density 95/Discs Large/Zonula Occludens(PDZ)-2 domain of Zonula Occludens-1 (ZO-1). The binding of the peptideto specific Cx43 C-terminus interaction domains such as PDZ-2 on ZO-1serves to competitively inhibit its association with the Cx43C-terminus. Connexins are gap junction proteins where as ZO-1 is a tightjunction associated protein. The binding of the 25 amino acid ACT1peptide to the PDZ-2 domain of ZO-1 serves to stabilize gap junctions aswell as tight junctions [12]. This junctional stability has beenassociated with faster healing and reduced scarring. In addition, thelow molecular weight of 25 amino acid peptide is expected to reduce therisk of an immunogenic response.

Granexin™ Gel is also designed to provide a localized protective barrieragainst microbial colonization and a moist environment to promotenatural autolytic debridement of necrotic tissue surrounding the wound.The lot number of Granexin™ Gel used in this study was DP 1493.

Central block randomization (Block size of 2) list was prepared by MNIindependent Biostatistician using a validated computer program(Statistical Analysis Software [SAS] 9.1.3). Subjects were randomized1:1 to either Group A (Granexin™ Gel plus SoC) or Group B (SoC only).Randomization was further stratified by wound size (<10 cm² and >10 cm²)to avoid bias of undue number of subjects with small or large woundsgoing into one arm of the study.

Interactive web response system (IWRS) was used for treatment groupassignment of the eligible subjects. Being the double-blinded study, anunblinded coordinator designated by the Investigator received theassigned treatment through IWRS.

Two studies, including a preclinical and clinical study tested theefficacy and safety of 100 μM concentration of ACT1 formulated inGranexin™ Gel. In the preclinical study, the efficacy of Granexin™ Gelin wound healing was assessed on 5 mm diameter full thickness excisionalwounds in adult mice and pigs. Wounds treated with 100 μM of ACT1peptide closed faster, appeared less swollen, inflamed and healed with asmoother and less discolored appearance as compared to the woundsapplied with the vehicle control. Subsequently, a Phase I, double blind,single-center, controlled study was conducted to evaluate the safety andtolerability of 20, 50, 100, and 200 μM concentrations of ACT1formulated in Granexin™ Gel versus placebo in 48 healthy subjectsfollowing punch biopsy. The results showed Granexin™ Gel (ACT1) at eachconcentration to be safe and well tolerated with no evidence oflocalized or systemic AEs or Serious Adverse Events (SAEs). Based onpreclinical studies and Phase I clinical trial, Granexin™ Gel with 100μM concentration of the ACT1 peptide was chosen for Phase II evaluationin the treatment of VLUs.

For each subject, Granexin™ Gel (100 μM ACT1 peptide) was applied atDays 0 and 3, and Weeks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12.

A total of 101 subjects with VLU were screened. Of these, 5 (4.9%) werescreen failures and 4 (4.04%) subjects fulfilled the inclusion/exclusioncriteria but did not come for the Baseline visit. The remaining 92(91.1%) subjects were enrolled and randomized in the study. A total of15 (16.3%) subjects met exclusion criteria number 1 or did not haveadequate evaluable wound photographic data post randomization and wereexcluded from the mITT and PP population.

A total of 70 (76.1%) subjects completed the study and the remaining 22(23.9%) subjects did not complete the study of which 14 (63.6%) withdrewtheir consent, 4 (18.2%) showed protocol non-compliance, 3 (13.6%) werelost to follow up, and 1 (4.5%) subject (Subject 1115; SoC group)reported death due to Myocardial infarction (Table 30. Table 30

Disposition of Subjects

Treatment Group Granexin ™ Gel plus SoC SoC Overall Category (N = 46) (N= 46) (N = 92) Population, n (%)^([1]) Screened 101 Randomized 46(100.0%) 46 (100.0%) 92 (100.0%) Intent-to-treat 46 (100.0%) 46 (100.0%)92 (100.0%) Modified Intent-to-treat 41 (89.1%) 36 (78.3%) 77 (83.7%)Per-Protocol 35 (76.1%) 33 (71.7%) 68 (73.9%) Safety 46 (100.0%) 46(100.0%) 92 (100.0%) Total number of subjects 34 (73.9%) 36 (78.3%) 70(76.1%) completed the study Total number of subjects 12 (26.1%) 10(21.7%) 22 (23.9%) not completed the study Reason for discontinuationConsent Withdrawn 10 (83.3%) 4 (40.0%) 14 (63.6%) Death  0 (0.0%)  1(10.0%)  1 (4.5%) Lost to follow up  2 (16.7%)  1 (10.0%)  3 (13.6%)Protocol non-compliance  0 (0.0%)  4 (40.0%)  4 (18.2%) Note:^([1])Percentage was calculated taking respective column header groupcount as denominator.

The following data sets were analyzed:

ITT Population: The ITT population included all subjects randomized toany treatment group, received at least one dose of study medication orreference treatment, and performed study assessments within and/oroutside the time window specified in the protocol.mITT Population: The mITT population excluded all subjects that metexclusion criterion 1 or did not have adequate evaluable woundphotographic data post randomizationPP Analysis Population: The PP analysis population was redefined toinclude subjects who did not have any major protocol violation.Safety Population: The safety population included all subjects who wererandomized to either of the treatment groups in the study.

The ITT population included 92 subjects; the mITT population included 77subjects, PP population included 68 subjects; and the safety populationincluded 92 subjects.

Modified Intent-to-Treat Population: The following subjects wereexcluded from the mITT population:

Granexin™ Gel plus SoC group: Subject 304, Subject 501, Subject 503,Subject 505, and Subject 906SoC group: Subject 103, Subject 104, Subject 1004, Subject 1006, Subject1008, Subject 1017, Subject 1022, Subject 1026, Subject 1027, andSubject 1031

Per Protocol Population: The following subjects were excluded from thePP population:

Meeting exclusion criterion—Decrease or increase in the ulcer size by30% or more during 7 day screening period or did not have adequateevaluable wound photographic data post randomization (15 subjects)

Granexin™ Gel plus SoC group: Subject 304, Subject 501, Subject 503,Subject 505, and Subject 906SoC group: Subject 103, Subject 104, Subject 1004, Subject 1006, Subject1008, Subject 1017, Subject 1022, Subject 1026, Subject 1027, andSubject 1031

Wound photograph data not available (9 subjects)

Granexin™ Gel plus SoC group: Subject 303, Subject 904, Subject 908,Subject 914, Subject 917, and subject 1102SoC group: Subject 502, Subject 1109, and Subject 1116

Demographics and Baseline Characteristics

Of 92 enrolled subjects, 83 (90.2%) were males and 9 (9.8%) werefemales. The mean±SD age was 49.8±12.71 years, weight was 70.47±14.89kg, height 168.35±7.19 cm, and BMI was 24.79±4.55 kg/m², respectively.Ten (10.9%) subjects had a smoking habit with the mean±SD number ofcigarettes smoked per day as 8.8±7.33.

TABLE 31 Summary of Subject Demographics at Screening Treatment GroupGranexin ™ Statistics/Category, Gel plus SoC SoC Overall n (%)^([1]) (N= 46) (N = 46) (N = 92) Age (years)^([2]) n 46 46 92 Mean 48.2 51.5 49.8SD 12.47 12.87 12.71 Median 48.0 53.0 51.0 Range (Min.:Max.) (18:71)(22:79) (18:79) Gender Male 44 39 83 (95.7) (84.8) (90.2%) Female 2 7 9(4.3) (15.2) (9.8%) Weight (kg)^([3]) n 46 46 92 Mean 72.15 68.79 70.47SD 15.014 14.739 14.891 Median 69.40 67.60 69.00 Range (Min.:Max.) (48.0:118.0)  (40.6:110.0)  (40.6:118.0) Height (cm)^([4]) n 46 46 92Mean 169.38 167.32 168.35 SD 5.918 8.223 7.199 Median 171.00 168.00169.85 Range (Min.:Max.) (156.0:180.0) (148.0:186.2) (148.0:186.2) BodyMass Index (kg/m²)^([5]) n 46 46 92 Mean 25.114 24.478 24.796 SD 4.75534.3794 4.5572 Median 23.930 23.807 23.807 Range (Min.:Max.)(16.04:38.97) (16.06:34.94) (16.04:38.97) Smoking Habits Yes 4 6 10(8.7) (13.0) (10.9%) No 42 40 82 (91.3) (87.0%) (89.1%) If yes, Current3 5 8 (6.5) (10.9) (8.7%) Past 1 1 2 (2.2) (2.2) (2.2%) Duration ofSmoking (months) n 4 6 10 Mean 300.0 345.7 327.4 SD 48.99 53.52 54.29Median 300.0 330.0 300.0 Range (Min.:Max.) (240:360) (300:420) (240:420)Missing 0 0 0 No. of Cigarettes Smoked per day n 3 5 8 Mean 8.8 8.8 8.8SD 9.67 6.87 7.33 Median 3.5 6.0 6.0 Range (Min.:Max.)  (3:20)  (2:20) (2:20) Missing 1 1 2 Note: ^([1])Percentages are calculated takingrespective column header group count as denominator. ^([2])Calculatedage as: Age = [(Informed consent signed Date (Screening) − Date ofBirth + 1)/365.25]. ^([3])Calculated Weight as: lbs/2.2 = kilograms.^([4])Calculated Height as: (Height (cms) = Height in inches * 2.538).^([5])Calculated BMI as: {Weight (kg)/(Height (m))²}.

Ulcer Identification History

Thirty (32.6%) subjects reported wound location on medial side of legfollowed by 20 (21.7%) subjects each reporting on anterior side of legand lateral side of leg. The overall mean±SD duration of wound historywas 68.4±149.65 weeks and mean wound area was 353.64±367.196 mm²,respectively (Table 32).

TABLE 32 Summary of Ulcer Identification History Treatment GroupGranexin ™ Statistics/Category, Gel plus SoC SoC Overall n (%)^([1]) (n= 46) (n = 46) (N = 92) Wound Location Anteromedial 0 (0.0) 1 (2.2)  1(1.1) Anterior side of leg 11 (23.9) 9 (19.6) 20 (21.7) LateralMalleolus 2 (4.3) 6 (13.0) 8 (8.7) Lateral Side of leg 12 (26.1) 8(17.4) 20 (21.7) Medial Malleolus 3 (6.5) 5 (10.9) 8 (8.7) Medial sideof leg 17 (37.0) 13 (28.3)  30 (32.6) Posterior side of leg 1 (2.2) 4(8.7)  5 (5.4) Duration of Wound History (weeks) n 46 46 92 Mean 62.774.0 68.4 SD 119.46 175.92 149.65 Median 16.0 16.0 16.0 Range(Min.:Max.) (4:511) (4:788)  (4:788)  Wound Area (mm²) (Length * Width)n 41 43 84 Mean 347.47 359.51 353.64 SD 369.266 369.484 367.196 Median236.43 233.43 234.93 Range (Min.:Max.) (19.0:1804.9) (0.0:1576.8)(0.0:1804.9) Note: ^([1])Percentages are calculated taking respectivecolumn header group count as denominator.

Primary Efficacy Endpoint: Mean Percent Wound Closure from Baseline toWeek 12

Table 33 summarizes the parametric analysis of the mean percentreduction of wound area (in mm²) from Baseline to Week 12 in the ITTpopulation. Of the 92 subjects in the ITT population, non-missingobservations from Baseline to Week 12 for percent reduction of woundarea were available for 62 subjects. The mean percent±SD reduction ofwound area from Baseline to Week 12 was not statistically significantlyhigher in Granexin™ Gel plus SoC group than SoC group (Granexin™ Gelplus SoC group: 79.28%±50.37; SoC group: 36.28%±179.78; p-value=0.0741)as per parametric analysis. Similar results were obtained aftersensitivity analysis (p-value=0.0921). The model factors such astreatment, visit, treatment*visit, and ankle circumference weresignificantly related to the mean percent reduction in wound area fromBaseline to Week 12 for the ITT population (Treatment: p-value=0.0062;Visit: p-value<0.0001; Treatment*Visit p-value=0.0139; AnkleCircumference: p-value=0.0200).

FIG. 10 displays the longitudinal response profile of the mean percentreduction of wound area at all visits by treatment group of the ITTpopulation.

TABLE 33 Mean Percent Reduction of Wound Area (mm²) from Baseline toWeek 12 (Parametric analysis) ITT Population (N = 92) Treatment GroupGranexin ™ Gel plus SoC SoC Statistics (N = 46) (N = 46) Total number ofsubjects evaluated N = 62^([1]) Percent reduction of wound area fromBaseline to Week 12 (mm²) n 34 28 Mean 79.279 36.282 SD 50.3695 179.7788Median 100.000 93.320 Range (Min.:Max.) (−68.85:100.00) (−849.50:100.00)LS Mean Estimate 73.2182 53.0164 Difference Estimate^([2]) 20.2018SE^([3]) 11.2336 95% CI (L:U.) (−1.99:42.39) p-value^([4],[5]) 0.0741Model factor^([4] [5]) p-value Treatment 0.0062 Visit <0.0001Treatment * Visit 0.0139 Exudate level 0.1440 Viable tissue 0.6703Strata 0.1532 Ankle circumference 0.0200 Wound duration 0.5371 BMI0.7303 Note: ^([1])“Total no. of subjects evaluated” representedsubjects having non-missing observations for change from Baseline toWeek 12 for percent reduction of wound area ^([2])Difference estimatefor Granexin ™ Gel plus SoC indicated (Granexin ™ Gel plus SoC)-SoC).^([3])SE of Granexin ™ Gel plus SoC indicated Standard Error ofDifferences (Granexin ™ Gel plus SoC)-SoC). ^([4])p-value for Granexin ™Gel plus SoC indicated significance of treatment differences (Granexin ™Gel plus SoC)-(SoC). ^([5])ANCOVA mixed model with repeated measures wasused to compare average percent reduction in wound area with treatment,visit and treatment * visit as factors and strata, wound duration,viable tissue, exudate level and the BMI as covariates using PROC Mixedprocedure of SAS software.

Table 34 summarizes the parametric analysis of the mean percentreduction of wound area (in mm²) from Baseline to Week 12 in the mITTpopulation. Of the 77 subjects in the mITT population, non-missingobservations from Baseline to Week 12 for percent reduction of woundarea were available for 59 subjects. The mean percent±SD reduction ofwound area from Baseline to Week 12 was not statistically significantlyhigher in Granexin™ Gel plus SoC group than SoC group (Granexin™ Gelplus SoC group: 78.96%±51.11; SoC group: 31.38%±185.89; p-value=0.0643)as per parametric analysis. Similar results were obtained aftersensitivity analysis (p-value=0.0785). The model factors such astreatment, visit, treatment*visit, and ankle circumference weresignificantly related to the mean percent reduction in wound area fromBaseline to Week 12 for the mITT population (Treatment: p-value=0.0028;Visit: p-value<0.0001; Treatment*Visit p-value=0.0424; AnkleCircumference: p-value=0.0428).

FIG. 11 displays the longitudinal response profile of the mean percentreduction of wound area at all visits by treatment group of the mITTpopulation.

TABLE 34 Mean Percent Reduction of Wound Area (mm²) from Baseline toWeek 12 (Parametric analysis) mITT Population (N = 77) Treatment GroupGranexin ™ Gel plus SoC SoC Statistics (N = 41) (N = 36) Total number ofsubjects evaluated N = 59^([1]) Percent reduction of wound area fromBaseline to Week 12 (mm²) n 33 26 Mean 78.957 31.380 SD 51.1148 185.8931Median 100.000 87.352 Range (Min.:Max.) (−68.85:100.00) (−849.50:100.00)LS Mean Estimate 73.275 51.921 Difference Estimate^([2]) 21.3543SE^([3]) 11.4565 95% CI (L:U.) (−1.28:43.99) p-value^([4],[5]) 0.0643Model factor^([4] [5]) p-value Treatment 0.0028 Visit <.0001 Treatment *Visit 0.0424 Exudate level 0.1232 Viable tissue 0.7318 Strata 0.1592Ankle circumference 0.0428 Wound duration 0.5846 BMI 0.6308 Note:^([1])“Total no. of subjects evaluated” represented subjects havingnon-missing observations for change from Baseline to Week 12 for percentreduction of wound area ^([2])Difference estimate for Granexin ™ Gelplus SoC indicated (Granexin ™ Gel plus SoC)-SoC). ^([3])SE ofGranexin ™ Gel plus SoC indicated Standard Error of Differences(Granexin ™ Gel plus SoC)-SoC). ^([4])p-value for Granexin ™ Gel plusSoC indicated significance of treatment differences (Granexin ™ Gel plusSoC)-(SoC). ^([5])ANCOVA mixed model with repeated measures was used tocompare average percent reduction in wound area with treatment, visitand treatment * visit as factors and strata, wound duration, viabletissue, exudate level and the BMI as covariates using PROC Mixedprocedure of SAS software.

The parametric analysis of the mean percent reduction of wound area (inmm²) from Baseline to Week 12 in the PP population is summarized inTable 35 Of the 68 subjects in the PP population, non-missingobservations for percent reduction of wound area from Baseline to Week12 were available for 58 subjects. The mean percent±SD reduction ofwound area from Baseline to Week 12 was not statistically significantlyhigher in Granexin™ Gel plus SoC group than in SoC group (Granexin™ Gelplus SoC group: 78.96%±51.11; SoC group: 28.63%±189.18) (p-value=0.0722)as per parametric analysis. Similarly, on applying LOCF approach forimputation of missing values, results obtained after sensitivityanalysis, were not statistically significant (p-value=0.1151. The modelfactors such as treatment, visit, treatment*visit, and anklecircumference were significantly related to the mean percent reductionin wound area from Baseline to Week 12 for the PP population (Treatment:p-value=0.0025; Visit: p-value<0.0001; Treatment*visit p-value=0.0228;and Ankle Circumference p-value=0.0350). FIG. 12 displays thelongitudinal response profile of the mean percentage reduction of woundarea at all visits by treatment group of the PP population.

TABLE 35 Mean Percent Reduction of Wound Area (mm²) from Baseline toWeek 12 (Parametric analysis) PP Population (N = 68) Treatment GroupGranexin ™ Gel plus SoC SoC Statistics (N = 35) (N = 33) Total no. ofsubjects evaluated N = 58^([1]) Percent reduction of wound area fromBaseline to Week 12 n 33 25 Mean 78.957 28.635 SD 51.1148 189.1879Median 100.000 85.225 Range (Min.:Max.) (−68.85:100.00) (−849.50:100.00)LS mean estimate 74.5294 53.5060 Difference estimate^([2]) 21.0234SE^([3]) 11.6048 95% CI (L.:U.) (−1.92:43.97) p-value^([4],[5]) 0.0722Model factor^([3][4]) p-value Treatment 0.0025 Visit <0.0001 Treatment *Visit 0.0228 Exudate level 0.1306 Viable tissue 0.7206 Strata 0.2849Ankle circumference 0.0350 Wound duration 0.5391 Body mass index 0.5793Note: ^([1])“Total no. of subjects evaluated” represented subjectshaving non-missing observations for dependent variable ^([2])Differenceestimate for Granexin ™ Gel plus SoC indicated (Granexin ™ Gel plusSoC) - SoC). ^([3])SE of Granexin ™ Gel plus Standard of Care indicatedStandard Error of Differences (Granexin ™ Gel plus SoC)- SoC).^([4])p-value for Granexin ™ Gel plus SoC indicated significance oftreatment differences (Granexin ™ Gel plus SoC) - (SoC). ^([5])ANCOVAmixed model with repeated measures was used to compare averagepercentage reduction in wound area with treatment, visit and treatment *visit as factors and strata, wound duration, viable tissue, exudatelevel and the body mass index as covariates using PROC Mixed procedureof SAS software.

To confirm the applicability of parametric statistical inferencemethodology in the wound reduction analysis, the data was tested todetermine if it followed normal distribution. Using PROC UNIVARIATE ofSAS® (version 9.1.3) Shapiro-Wilk W statistic was calculated. Thep-value (<0.0001) indicated that the data did not follow a normaldistribution. This was further confirmed by the Q-Q plot for both theITT and PP populations in which the observations were not in a straightline and did not show a normal distribution. Therefore, the hypothesisof normal distribution of the data in the sample was rejected andnon-parametric analysis was conducted.

Table 36 summarizes the non-parametric analysis for mean percentreduction of wound area from Baseline to Week 12 in the ITT population.The Wilcoxon Mann-Whitney U test is a non-parametric test whichdetermines whether there is a difference in two samples of independentobservations. In Granexin™ Gel plus SoC group, the actual sum of scores(1210.00) was found higher than the expected sum of scores under thenull hypothesis (1071.00). In SoC group, the actual sum of scores(743.00) was found lower than the expected sum of the scores under thenull hypothesis (882.00). The actual sum of scores was calculated byadding the ranks of both groups after sorting data of wound sizereduction in ascending order and the expected sum of scores wascalculated as the sum of scores when there is no difference between the2 groups. Subjects in Granexin™ Gel plus SoC group had statisticallysignificantly higher percent reduction of wound area from Baseline toWeek 12 than in SoC group (p-value=0.0238).

TABLE 36 Mean Percent Reduction of Wound Area (mm²) from Baseline toWeek 12 (Non-parametric analysis) ITT Population (N = 92) TreatmentGroup Granexin ™ Gel plus SoC SoC Statistics (N = 46) (N = 46) Percentreduction of wound area from Baseline to Week 12 Sum of score 1210.00743.00 Expected under H_(o) 1071.00 882.00 p-value^([1]) 0.0238 Note:^([1])p-value is evaluated by using non-parametric Wilcoxon Mann-WhitneyU test between treatment group. General Note: Mean percentage reductionfrom Baseline = ((Baseline − week X)/Baseline) * 100 H_(o): NullHypothesis If wound was healed and all further visits were missing then100% reduction had been carried forward to missing visits.

Table 37 summarizes the non-parametric analysis for mean percentreduction of wound area from Baseline to Week 12 in the mITT population.The Wilcoxon Mann-Whitney U test is a non-parametric test whichdetermines whether there is a difference in two samples of independentobservations. In Granexin™ Gel plus SoC group, the actual sum of scores(1136.00) was found higher than the expected sum of scores under thenull hypothesis (990.00). In SoC group, the actual sum of scores(634.00) was found lower than the expected sum of the scores under thenull hypothesis (780.00). The actual sum of scores was calculated byadding the ranks of both groups after sorting data of wound sizereduction in ascending order and the expected sum of scores wascalculated as the sum of scores when there is no difference between the2 groups. Subjects in Granexin™ Gel plus SoC group had statisticallysignificantly higher percent reduction of wound area from Baseline toWeek 12 than in SoC group (p-value=0.0105).

TABLE 37 Mean Percent Reduction of Wound Area (mm²) from Baseline toWeek 12 (Non-parametric analysis) mITT Population (N = 77) TreatmentGroup Granexin ™ Gel plus SoC SoC Statistics (N = 41) (N = 36) Percentreduction of wound area from Baseline to Week 12 Sum of score 1136.00634.00 Expected under H_(o) 990.00 780.00 p-value^([1]) 0.0105 Note:^([1])p-value is evaluated by using non-parametric Wilcoxon Mann-WhitneyU test between treatment group. General Note: Mean percentage reductionfrom Baseline = ((Baseline − week X)/Baseline) * 100 H_(o): NullHypothesis If wound was healed and all further visits were missing then100% reduction had been carried forward to missing visits.

Table 38 summarizes the non-parametric analysis for mean percentreduction of wound area from Baseline to Week 12 in the PP population.In Granexin™ Gel plus SoC group, the actual sum of scores (1123.00) wasfound higher than the expected sum of scores under the null hypothesis(973.50) and in SoC group, the actual sum of scores (588.00) was foundlower than the expected sum of the scores under the null hypothesis(737.50). Subjects in Granexin™ Gel plus SoC group had statisticallysignificantly higher percent reduction of wound area from Baseline toWeek 12 than subjects in SoC group (p-value=0.0073).

TABLE 38 Mean Percent Reduction of Wound Area (mm²) from Baseline toWeek 12 (Non-parametric analysis) PP Population (N = 68) Treatment GroupGranexin ™ Gel plus SoC group SoC group Statistics (N = 35) (N = 33)Percent reduction of wound area from Baseline to Week 12 Sum of score1123.00 588.00 Expected under H_(o) 973.50 737.50 p-value^([1]) 0.0073Note: ^([1])p-value was evaluated by using non-parametric WilcoxonMann-Whitney U test between treatment group.

Secondary Analysis: Mean Percentage Reduction of Wound Area fromBaseline to Week 4

The parametric evaluation of mean percent wound closure from Baseline toWeek 4 in the ITT population is summarized in Table 39. Of 92 subjectsin the ITT population, non-missing observations for percent reduction ofwound area from Baseline to Week 4 were available for a total of 56subjects. The mean percent±SD reduction of wound area from Baseline toWeek 4 was statistically significantly higher in Granexin™ Gel plus SoCgroup (76.99±29.74) than in SoC group (39.080±42.80) (p-value=0.0040).The model factors such as treatment, visit, and ankle circumference weresignificantly related to the mean percent reduction of wound area fromBaseline to Week 4 in the ITT population (treatment p-value=0.0062;Visit p-value<0.0001; treatment*visit p-value=0.0139; and AnkleCircumference p-value=0.0200).

TABLE 39 Mean Percent Reduction of Wound Area (mm²) from Baseline toWeek 4 (Parametric analysis) ITT Population (N = 92) Treatment GroupGranexin ™ Gel plus SoC SoC Statistics (N = 46) (N = 46) Total no. ofsubjects evaluated N = 56^([1]) Percent reduction of wound area fromBaseline to Week 4 n 30 26 Mean 76.996 39.080 SD 29.7470 42.8061 Median97.369 41.872 Range (Min.:Max.)  (0.40:100.00) (−35.54:100.00) LS meanestimate 60.2593 27.0027 Difference estimate [2] 33.2566 SE[3] 11.388395% CI (L.:U.) (10.76:55.75) p-value [4], [5] 0.0040 ModelFactor^([3][4]) p-value Treatment 0.0062 Visit <0.0001 Treatment * Visit0.0139 Exudate level 0.1440 Viable tissue 0.6703 Strata 0.1532 AnkleCircumference 0.0200 Wound duration 0.5371 Body mass index 0.7303 Note:^([1])“Total no. of subjects evaluated” represented subjects havingnon-missing observations for dependent variable [2] Difference estimatefor Granexin ™ Gel plus SoC indicated (Granexin ™ Gel plus SoC) - SoC).^([3])SE of Granexin ™ Gel plus SoC indicated Standard Error ofDifferences (Granexin ™ Gel plus SoC)- SoC). ^([4])p-value forGranexin ™ Gel plus SoC indicated significance of treatment differences(Granexin ™ Gel plus SoC) - (SoC). [5] ANCOVA mixed model with repeatedmeasures was used to compare average percentage reduction in wound areawith treatment, visit and treatment * visit as factors and strata, woundduration, viable tissue, exudate level and the body mass index ascovariates using PROC Mixed procedure of SAS software.

The parametric evaluation of mean percent wound closure from Baseline toWeek 4 in the mITT population is summarized in Table 40. Of 77 subjectsin the mITT population, non-missing observations for percent reductionof wound area from Baseline to Week 4 were available for a total of 54subjects. The mean percent±SD reduction of wound area from Baseline toWeek 4 was statistically significantly higher in Granexin™ Gel plus SoCgroup (76.99±29.74) than in SoC group (34.00±40.52) (p-value=0.0022).The model factors such as treatment, visit, and ankle circumference weresignificantly related to the mean percent reduction of wound area fromBaseline to Week 4 in the mITT population (treatment p-value=0.0028;Visit p-value<0.0001; treatment*visit p-value=0.0424; and AnkleCircumference p-value=0.0428).

TABLE 40 Mean Percent Reduction of Wound Area (mm²) from Baseline toWeek 4 (Parametric analysis) mITT Population (N = 77) Treatment GroupGranexin ™ Gel plus SoC Statistics SoC (N = 41) (N = 36) Total no. ofsubjects evaluated N = 54^([1]) Percent reduction of wound area fromBaseline to Week 4 n 30 24 Mean 76.996 34.003 SD 29.7470 40.5227 Median97.369 38.217 Range (Min.:Max.)  (0.40:100.00) (−35.54:100.00) LS meanestimate 60.394 24.421 Difference estimate [2] 35.9729 SE[3] 11.5191 95%CI (L.:U.) (13.21:58.73) p-value [4], [5] 0.0022 Model Factor^([3][4])p-value Treatment 0.0028 Visit <0.0001 Treatment * Visit 0.0424 Exudatelevel 0.1232 Viable tissue 0.7318 Strata 0.1592 Ankle Circumference0.0428 Wound duration 0.5846 Body mass index 0.6308 Note: ^([1])“Totalno. of subjects evaluated” represented subjects having non-missingobservations for dependent variable [2] Difference estimate forGranexin ™ Gel plus SoC indicated (Granexin ™ Gel plus SoC) - SoC).^([3])SE of Granexin ™ Gel plus SoC indicated Standard Error ofDifferences (Granexin ™ Gel plus SoC)- SoC). ^([4])p-value forGranexin ™ Gel plus SoC indicated significance of treatment differences(Granexin ™ Gel plus SoC) - (SoC). [5] ANCOVA mixed model with repeatedmeasures was used to compare average percentage reduction in wound areawith treatment, visit and treatment * visit as factors and strata, woundduration, viable tissue, exudate level and the body mass index ascovariates using PROC Mixed procedure of SAS software.

The parametric analysis of mean percentage reduction of wound area fromBaseline to Week 4 in the PP population is summarized in Table 41. Of 68subjects in the PP population, non-missing observations for change fromBaseline to Week 4 for percent reduction of wound area were availablefor a total of 53 subjects. The mean percent±SD reduction of wound area(mm²) from Baseline to Week 4 was statistically significantly higher inGranexin™ Gel plus SoC group subjects (76.99±29.74) than in SoC groupsubjects (31.13±38.86) (p-value=0.0012). The model factors such astreatment, visit, treatment*visit, and ankle circumference weresignificantly related to the mean percent reduction of wound area fromBaseline to Week 4 in the PP population (treatment p-value=0.0025; Visitp-value<0.0001; treatment*visit p-value=0.0228; and Ankle Circumferencep-value=0.0350).

TABLE 41 Mean Percent Reduction of Wound Area (mm²) from Baseline toWeek 4 (Parametric analysis) PP Population (N = 68) Treatment GroupGranexin ™ Gel plus SoC Statistics SoC (N = 35) (N = 33) Total no. ofsubjects evaluated N = 53^([1]) Percent reduction of wound area fromBaseline to Week 4 n 30 23 Mean 76.996 31.134 SD 29.7470 38.8605 Median97.369 34.902 Range (Min.:Max.)  (0.40:100.00) (−35.54:100.00) LS MeanEstimate 62.3396 23.8304 Difference Estimate^([2]) 38.5092 SE^([3])11.6027 95% CI (L.:U.) (15.56:61.46) p-value^([4],[5]) 0.0012 ModelFactor^([3][4]) p-value Treatment 0.0025 Visit <0.0001 Treatment * Visit0.0228 Exudate level 0.1306 Viable tissue 0.7206 Strata 0.2849 Anklecircumference 0.0350 Wound duration 0.5391 Body mass index 0.5793 Note:^([1])“Total no. of subjects evaluated” represented subjects havingnon-missing observations for dependent variable ^([2])Differenceestimate for Granexin ™ Gel plus SoC indicated (Granexin ™ Gel plusSoC) - SoC). ^([3])SE of Granexin ™ Gel plus SoC indicated StandardError of Differences (Granexin ™ Gel plus SoC)- SoC). ^([4])p-value forGranexin ™ Gel plus SoC indicated significance of treatment differences(Granexin ™ Gel plus SoC) - (SoC). ^([5])ANCOVA mixed model withrepeated measures was used to compare average percentage reduction inwound area with treatment, visit and treatment * visit as factors andstrata, wound duration, viable tissue, exudate level and the body massindex as covariates using PROC Mixed procedure of SAS software.

Table 42 summarizes the non-parametric analysis for mean percentreduction of wound area from Baseline to Week 4 in the ITT population.In Granexin™ Gel plus SoC group, the actual sum of scores (1060.00) wasfound higher than the expected sum of scores under the null hypothesis(855.00). In SoC group, the actual sum of scores (536.00) was foundlower than the expected sum of the scores under the null hypothesis(741.00). Granexin™ Gel plus SoC group subjects had statisticallysignificantly higher percent reduction of wound area than SoC groupsubjects from Baseline to Week 4 (p-value=0.0006).

TABLE 42 Mean Percent Reduction of Wound Area (mm²) from Baseline toWeek 4 (Non-parametric analysis) ITT Population (N = 92) Treatment GroupGranexin ™ Gel plus SoC SoC Statistics (N = 46) (N = 46) Percentreduction of wound area from Baseline to Week 4 Sum of score 1060.00536.00 Expected under H_(o) 855.00 741.00 p-value^([1]) 0.0006 Note:^([1])p-value is evaluated by using non-parametric Wilcoxon Mann-WhitneyU test between treatment group.

Table 43 summarizes the non-parametric analysis for mean percentreduction of wound area from Baseline to Week 4 in the mITT population.In Granexin™ Gel plus SoC group, the actual sum of scores (1046.00) wasfound higher than the expected sum of scores under the null hypothesis(825.00). In SoC group, the actual sum of scores (439.00) was foundlower than the expected sum of the scores under the null hypothesis(660.00). Granexin™ Gel plus SoC group subjects had statisticallysignificantly higher percent reduction of wound area than SoC groupsubjects from Baseline to Week 4 (p-value=0.0001).

TABLE 43 Mean Percent Reduction of Wound Area (mm²) from Baseline toWeek 4 (Non-parametric analysis) mITT Population (N = 77) TreatmentGroup Granexin ™ Gel plus SoC SoC Statistics (N = 41) (N = 36) Percentreduction of wound area from Baseline to Week 4 Sum of score 1046.00439.00 Expected under H_(o) 825.00 660.00 p-value^([1]) 0.0001 Note:^([1])p-value is evaluated by using non-parametric Wilcoxon Mann-WhitneyU test between treatment group.

The non parametric analysis for mean percent reduction of wound areafrom Baseline to Week 4 in the PP population is summarized in Table 44.In Granexin™ Gel plus SoC group, the actual sum of scores (1039.00) wasfound higher than the expected sum of scores under the null hypothesis(810.00). In SoC group, the actual sum of scores (392.00) was foundlower than the expected sum of the scores under the null hypothesis(621.00). Granexin™ Gel plus SoC group subjects had statisticallysignificantly higher percent reduction of wound area than SoC groupsubjects from Baseline to Week 4 (p-value<0.0001).

TABLE 44 Mean Percent Reduction of Wound Area (mm²) from Baseline toWeek 4 (Non-parametric analysis) PP Population (N = 68) Treatment GroupGranexin ™ Gel plus SoC SoC Statistics (N = 35) (N = 33) Percentreduction of wound area from Baseline to Week 4 Sum of score 1039.00392.00 Expected under H_(o) 810.00 621.00 p-value^([1]) <0.0001 Note:^([1])p-value is evaluated by using non-parametric Wilcoxon Mann-WhitneyU test between treatment group.

Time to 100% Wound Closure: Time taken for first complete (100%) woundclosure for the ITT population is summarized in Table 45 and FIG. 13Complete wound closure was defined as 100% epithelialization of thewound and absence of any drainage from the wound. Out of 92 subjects, 28[60.9%] subjects in the Granexin™ Gel plus SoC group and 15 (32.6%)subjects in the SoC group had 100% wound closure by Week 12. The medianduration of 100% wound closure was 6 weeks (90% CI: 4 to 8 weeks) inGranexin™ Gel plus SoC group and 12.14 weeks (90% CI: 10.14 to notachieved) in SoC group. Subjects in Granexin™ Gel plus SoC group tookstatistically significantly less time to achieve 100% wound closure thanSoC group subjects (p-value=0.0006). Five subjects (Granexin™ Gel plusSoC group: 4; SoC group: 1) having missing value after Screening orhaving missing visit date were not included in the analysis. Theremaining 44 subjects (Granexin™ Gel plus SoC group: 14 [30.4%]; SoCgroup: 30 [65.2%]) were censored due to reasons such as subjects nothaving complete wound closure, lack of photographic evaluation data,withdrawal of consent, loss to follow-up, discontinued due to protocolnon compliance, or death.

TABLE 45 Summary of Time to First Complete (100%) Wound Closure by Week12 ITT Population (N = 92) Treatment Group Granexin ™ Gel plus SoC SoCStatistics^([1]) (N = 46) (N = 46) Number (%) of subjects 28 (60.9%) 15(32.6%) with complete (100%) wound closure Number (%) of censored 14(30.4%) 30 (65.2%) subjects Median duration (weeks) 6.00 (4.00; 8.00)12.14 (10.14; NA) of complete wound closure(90% CI)^([2]) p-value^([3])0.0006 Note: ^([1])Percentage was calculated taking count ofcorresponding treatment groups as denominator. ^([2])The median durationof complete wound closure was estimated by Kaplan-Meier method and also90% CI was calculated for the median duration of 100% wound closure.^([3])p-value was calculated using log-rank test for treatment groupsusing Proc Lifetest procedure of SAS software.

Time taken for first complete (100%) wound closure for the mITTpopulation is summarized in Table 46 and FIG. 14Error! Reference sourcenot found. Complete wound closure was defined as 100% epithelializationof the wound and absence of any drainage from the wound. Out of 77subjects, 28 (68.3%) subjects in the Granexin™ Gel plus SoC group and 15(36.1%) subjects in the SoC group had 100% wound closure by Week 12. Themedian duration of 100% wound closure was 6 weeks (90% CI: 4 to 7 weeks)in Granexin™ Gel plus SoC group and not achieved (90% CI: 12 to notachieved) in SoC group. Subjects in Granexin™ Gel plus SoC group tookstatistically significantly less time to achieve 100% wound closure thanSoC group subjects (p-value<0.0001). Five subjects (Granexin™ Gel plusSoC group: 4; SoC group: 1) having missing value after Screening orhaving missing visit date were not included in the analysis. Theremaining 34 subjects (Granexin™ Gel plus SoC group: 11 (26.8%); SoCgroup: 23 (63.9%) were censored due to reasons such as subjects nothaving complete wound closure, lack of photographic evaluation data,withdrawal of consent, loss to follow-up, discontinued due to protocolnon compliance, or death.

TABLE 46 Summary of Time to First Complete (100%) Wound Closure by Week12 mITT Population (N = 77) Treatment Group Granexin ™ Gel plus SoC SoCStatistics^([1]) (N = 41) (N = 36) Number (%) of subjects 28 (68.3%) 13(36.1%) with complete (100%) wound closure Number (%) of censored 11(26.8%) 23 (63.9%) subjects Median duration (weeks) 6.00 (4.00; 7.00) NA(12.00; NA)^([3]) of complete wound closure(90% CI)^([2]) p-value^([4])<0.0001 Note: ^([1])Percentage was calculated taking count ofcorresponding treatment groups as denominator. ^([2])The median durationof complete wound closure was estimated by Kaplan-Meier method and also90% CI was calculated for the median duration of 100% wound closure.^([3])NA: Not achieved ^([4])p-value was calculated using log-rank testfor treatment groups using Proc Lifetest procedure of SAS software.

Time taken for 100% wound closure in the PP population is summarized inTable 47 and FIG. 15. Out of 68 subjects, 40 (Granexin™ Gel plus SoCgroup: 28 [80.0%]; SoC group: 12 [36.4%]) had 100% wound closure by Week12. The median duration of 100% wound closure was 6 weeks (90% CI was 4to 7 weeks) in Granexin™ Gel plus SoC group and median duration was notachieved (90% CI: 12.00 to NA) in 12 weeks of treatment evaluation inSoC group. Subjects in Granexin™ Gel plus SoC group took statisticallysignificantly less time to achieve 100% wound closure than SoC groupsubjects (p-value<0.0001). A total of 28 subjects (Granexin™ Gel plusSoC group: 7[20.0%]; SoC: 21 (63.6%]) were censored due to reasons suchas subjects not having complete wound 100% closure, lack of photographicevaluation data, withdrawal of consent, loss to follow-up, discontinueddue to protocol non compliance, or death.

TABLE 47 Summary of Time to First Complete (100%) Wound Closure by Week12 PP Population (N = 68) Treatment Group Granexin ™ Gel plus SoC SoCStatistics^([1]) (N = 35) (N = 33) Number (%) of subjects 28 (80.0%) 12(36.4%) with complete (100%) wound closure Number (%) of censored 7(20.0%) 21 (63.6%) subjects Median duration (weeks) 6.00 (4.00; 7.00) NA(12.00; NA) of complete wound closure (90% CI)^([2]) p-value^([3])<0.0001 Note: ^([1])Percentage was calculated taking count ofcorresponding treatment groups as denominator. ^([2])The median durationof complete wound closure was estimated by Kaplan-Meier method and also90% CI was calculated for the median duration of 100% wound closure.^([3])p-value was calculated using Log-rank test for treatment groupsusing Proc Lifetest procedure of SAS software.

Time taken to achieve 50% wound closure in the ITT population issummarized in Table 48 and FIG. 16. Out of 92 subjects, 61 (Granexin™Gel plus SoC group: 34 [73.9%]; SoC group: 25 [54.3%]) had 50% woundclosure by Week 12. The median duration of 50% wound closure was 2.86weeks (90% CI was 2.14 to 3 weeks) in Granexin™ Gel plus SoC group and6.86 weeks (90% CI: 5.00 to 9.14) in SoC group. Subjects in Granexin™Gel plus SoC group took statistically significantly less time to achieve50% wound closure than SoC group subjects (p-value=0.0002). Five (5.5%)subjects having missing values after Screening or having missing visitdates were not included in the analysis. A total of 28 subjects(Granexin™ Gel plus SoC group: 8 [17.4%]; SoC group: 20 [43.5%]) werecensored due to reasons such as subjects not having complete 50% woundclosure, lack of photographic evaluation data, withdrawal of consent,loss to follow-up, discontinued due to protocol non compliance, ordeath.

TABLE 48 Summary of Time to First Complete 50% Wound Closure by Week 12ITT Population (N = 92) Treatment Group Granexin ™ Gel plus SoC SoCStatistics^([1]) (n = 46) (n = 46) Number (%) of subjects 34 (73.9%) 25(54.3%) with complete (50%) wound closure Number (%) of censored 8(17.4%) 20 (43.5%) subjects Median duration (weeks) 2.86 (2.14; 3.00)6.86 (5.00; 9.14) of complete wound closure (90% CI)^([2]) p-value^([3])0.0002 Note: ^([1])Percentage was calculated taking count ofcorresponding treatment groups as denominator. ^([2])The median durationof complete wound closure was estimated by Kaplan-Meier method and also90% confidence interval was calculated for the median duration of 50%wound closure. ^([3])p-value was calculated using Log-rank test fortreatment groups using Proc Lifetest procedure of SAS software

Time taken to achieve 50% wound closure in the mITT population issummarized in Table 49 and FIG. 17. Out of 77 subjects, 59 (Granexin™Gel plus SoC group: 33 [80.5%]; SoC group: 23 [63.9%]) had 50% woundclosure by Week 12. The median duration of 50% wound closure was 2.86weeks (90% CI was 2.14 to 3 weeks) in Granexin™ Gel plus SoC group and 8weeks (90% CI: 5.14 to 9.86) in SoC group. Subjects in Granexin™ Gelplus SoC group took statistically significantly less time to achieve 50%wound closure than SoC group subjects (p-value<0.0001). Five (5.5%)subjects having missing values after Screening or having missing visitdates were not included in the analysis. A total of 19 subjects(Granexin™ Gel plus SoC group: 6 [14.6%]; SoC group: 13 [36.1%]) werecensored due to reasons such as subjects not having complete 50% woundclosure, lack of photographic evaluation data, withdrawal of consent,loss to follow-up, discontinued due to protocol non compliance, ordeath.

TABLE 49 Summary of Time to First Complete 50% Wound Closure by Week 12mITT Population (N = 77) Treatment Group Granexin ™ Gel plus SoC SoCStatistics^([1]) (n = 41) (n = 36) Number (%) of subjects with 33(80.5%) 23 (63.9%) complete (50%) wound closure Number (%) of censored 6(14.6%) 13 (36.1%) subjects Median duration (weeks) of 2.86 (2.00; 3.00)8.00 (5.14; 9.86) complete wound closure (90% CI)^([2]) p-value^([3])<0.0001 Note: ^([1])Percentage was calculated taking count ofcorresponding treatment groups as denominator. ^([2])The median durationof complete wound closure was estimated by Kaplan-Meier method and also90% confidence interval was calculated for the median duration of 50%wound closure. ^([3])p-value was calculated using Log-rank test fortreatment groups using Proc Lifetest procedure of SAS software.

The time taken to achieve 50% wound closure in the PP population issummarized in Table 50 and FIG. 18. Out of 68 subjects, 54 (Granexin™Gel plus SoC group: 32 [91.4%]; SoC group: 22 [66.7%]) had 50% woundclosure by Week 12. The median duration of 50% wound closure was 2.86weeks (90% CI was 2 to 3 weeks) in Granexin™ Gel plus SoC group and 8weeks (90% CI: 5.14 to 9.86) in SoC group. Subjects in Granexin™ Gelplus SoC group took statistically significantly less time to achieve 50%wound closure than SoC group subjects (p-value<0.0001). A total of 14subjects (Granexin™ Gel plus SoC group: 3 [8.6%]; SoC: 11 [33.3%]) werecensored as they did not complete 50% wound closure during the studyperiod, or due to lack of photographic evaluation data, withdrawal ofconsent, loss to follow-up, discontinued due to protocol non compliance,or death.

TABLE 50 Summary of Time to First Complete (50%) Wound Closure by Week12 PP Population (N = 68) Treatment Group Granexin ™ Gel plus SoC SoCStatistics^([1]) (N = 35) (N = 33) Number (%) of subjects with 32(91.4%) 22 (66.7%) complete (50%) wound closure Number (%) of censored 3(8.6%) 11 (33.3%) subjects Median duration (weeks) of 2.86 (2.00; 3.00)8.00 (5.14; 9.86) complete wound closure (90% CI)^([2]) p-value^([3])<0.0001 Note: ^([1])Percentage was calculated taking count ofcorresponding treatment groups as denominator. ^([2])The median durationof complete wound closure was estimated by Kaplan-Meier method and also90% CI was calculated for the median duration of 50% wound closure.^([3])p-value was calculated using Log-rank test for treatment groupsusing Proc Lifetest procedure of SAS software.

Additional to the Kaplan-Meier Model, Cox Proportional HazardsRegression Model was also used to compare the event-time distributionfunction between the two treatment groups. This analysis was conductedto evaluate if one or more covariates were associated with the time to100% or 50% wound closure. Table 51 below summarizes the CoxProportional Hazards Regression Analysis of time to complete woundclosure in the ITT population. The effect of covariates estimated by theproportional hazards model is reported as hazard ratios. As per thehazards ratio and p-value, covariates such as treatment group and woundsize showed an association with time to completion of wound closure(treatment group p-value=0.0406; wound size p-value=0.0265) and thesubjects treated with Granexin™ Gel plus SoC would have wound closure2.281 times more than the subjects treated with SoC only.

TABLE 51 Analysis of Time to First Complete (100%) Wound Closure (Cox'sProportional Hazards Regression Analysis)ITT Population (N = 92) Hazard95% CI for Hazard Ratio Statistics Ratio ^([1]) Lower Upper p-valueTreatment group 2.281 1.036 5.021 0.0406 Wound size 0.998 0.997 1.0000.0265 Wound duration 1.000 0.996 1.003 0.8431 Baseline wound depth1.767 0.312 10.006 0.5197 BMI 1.003 0.922 1.092 0.9423 Note: ^([1]) CoxProportional Hazard ratio with 95% CI and p-value was calculated usingPROC PHREG procedure.

Table 52 below summarizes the Cox Proportional Hazards RegressionAnalysis of time to complete wound closure in the mITT population. Theeffect of covariates estimated by the proportional hazards model isreported as hazard ratios. As per the hazards ratio and p-value,covariate such as treatment group showed an association with time tocompletion of wound closure (p-value=0.0139) and the subjects treatedwith Granexin™ Gel plus SoC would have wound closure 2.776 times morethan the subjects treated with SoC only. Wound size, wound duration,baseline wound depth, and BMI were not associated with time to woundclosure.

TABLE 52 Analysis of Time to First Complete (100%) Wound Closure (Cox'sProportional Hazards Regression Analysis) mITT Population (N = 77)Hazard 95% CI for Hazard Ratio Statistics Ratio ^([1]) Lower Upperp-value Treatment group 2.776 1.230 6.265 0.0139 Wound size 0.998 0.9971.000 0.0629 Wound duration 1.000 0.996 1.003 0.8838 Baseline wounddepth 2.306 0.409 13.004 0.3439 BMI 1.021 0.937 1.113 0.6386 Note:^([1]) Cox Proportional Hazard ratio with 95% CI and p-value wascalculated using PROC PHREG procedure.

Cox Proportional Hazards Regression Analysis for time to 100% woundclosure in the PP population is summarised in Table 53 below. The effectof covariates estimated by the proportional hazards model is reported ashazard ratios. As per the hazards ratio and p-value, covariates such astreatment group showed an association with time to completion of woundclosure (p-value=0.0077) and the subjects treated with Granexin™ Gelplus SoC would have wound closure 3.150 times more than the subjectstreated with SoC only. Wound size, wound duration, baseline wound depth,and BMI were not associated with time to wound closure.

TABLE 53 Analysis of Time to First Complete (100%) Wound Closure (Cox'sProportional Hazards Regression Analysis) PP Population (N = 68) Hazard95% CI for Hazard Ratio Statistics Ratio ^([1]) Lower Upper p-valueTreatment Group 3.150 1.354 7.326 0.0077 Wound size 0.999 0.997 1.0000.0714 Wound duration 1.000 0.997 1.003 0.9942 Baseline wound depth2.027 0.333 12.352 0.4435 BMI 1.021 0.935 1.114 0.6449 Note: ^([1]) CoxProportional Hazard ratio with 95% CI and p-value was calculated usingPROC PHREG procedure.

Time to 50% wound closure in the ITT population is summarized in Table54. As per the hazards ratio and p-value, covariates such as treatmentgroup and wound size, showed an association with time to completion ofwound closure (treatment group p-value=0.0143; wound sizep-value=0.0162) and the subjects treated with Granexin™ Gel plus SoCwould have wound closure 2.247 times more than the subjects treated withSoC only.

TABLE 54 Analysis of Time to First Complete (50%) Wound Closure (Cox'sProportional Hazards Regression Analysis) ITT Population (N = 92) Hazard95% CI for Hazard Ratio Statistics Ratio^([1]) Lower Upper p-valueTreatment Group 2.247 1.176 4.294 0.0143 Wound size 0.999 0.997 1.0000.0162 Wound duration 1.000 0.997 1.002 0.7702 Baseline wound depth0.680 0.182 2.537 0.5654 BMI 0.988 0.928 1.052 0.7046 Note: ^([1])CoxProportional Hazard ratio with 95% confidence interval and p-value wascalculated using PROC PHREG procedure.

Time to 50% wound closure in the mITT population is summarized in Table55. As per the hazards ratio and p-value, covariates such as treatmentgroup and wound size, showed an association with time to completion ofwound closure (treatment group p-value=0.0067; wound sizep-value=0.0346) and the subjects treated with Granexin™ Gel plus SoCwould have wound closure 2.501 times more than the subjects treated withSoC only.

TABLE 55 Analysis of Time to First Complete (50%) Wound Closure (Cox'sProportional Hazards Regression Analysis) mITT Population (N = 77)Hazard 95% CI for Hazard Ratio Statistics Ratio^([1]) Lower Upperp-value Treatment Group 2.501 1.289 4.853 0.0067 Wound size 0.999 0.9981.000 0.0346 Wound duration 1.000 0.997 1.002 0.8310 Baseline wounddepth 0.805 0.217 2.991 0.7461 BMI 0.998 0.937 1.064 0.9585 Note:^([1])Cox Proportional Hazard ratio with 95% confidence interval andp-value was calculated using PROC PHREG procedure.

Time to 50% wound closure in the PP population is summarized in Table56. As per the hazards ratio and p-value, covariates such as treatmentgroup and wound size, showed an association with time to completion of50% wound closure (treatment group p-value=0.0059; wound sizep-value=0.0442) and the subjects treated with Granexin™ Gel plus SoCwould have wound closure 2.611 times more than the subjects treated withSoC only.

TABLE 56 Analysis of Time to First Complete (50%) Wound Closure (Cox'sProportional Hazards Regression Analysis) PP Population (N = 68) Hazard95% CI for Hazard Ratio Statistics Ratio^([1]) Lower Upper p-valueTreatment Group 2.611 1.319 5.169 0.0059 Wound size 0.999 0.998 1.0000.0442 Wound duration 1.000 0.997 1.002 0.8788 Baseline wound depth0.604 0.151 2.412 0.4755 BMI 0.998 0.935 1.064 0.9469 Note: ^([1])CoxProportional Hazard ratio with 95% confidence interval and p-value wascalculated using PROC PHREG procedure.

Wound Recurrence

Overall in ITT, mITT, and PP population, wound recurrence was reportedin 10 subjects (Granexin™ Gel plus SoC group: 5 subjects; SoC group: 5subjects) for the entire duration of the study. Of these 10 subjects,wound recurrence was observed in 2 subjects in Granexin™ Gel plus SoCgroup (Subject 504, Subject 907) and 2 subjects in SoC group (Subject1003; Subject 1103) after completion of treatment at 12 weeks. There wasno statistically significant difference in wound recurrence between the2 treatment groups (p-value≧0.9999).

TABLE 57 Wound Recurrence in the Overall Population Treatment GroupGranexin ™ Gel plus SoC SoC Overall Recurrence, n (%)^([1]) (N = 46) (N= 46) (N = 92) At any visit Yes  5 (10.9%)  5 (10.9%) 10 (10.9%) No 41(89.1%) 41 (89.1%) 82 (89.1%) p-value^([2]) —     ≧0.9999  —     AfterWeek 12 Yes 2 (4.3%) 2 (4.3%) 4 (4.3%) No 44 (95.7%) 44 (95.7%) 88(95.7%) p-value^([2]) —     ≧0.9999  —     Note: ^([1])Percentage wascalculated by taking respective column header group count asdenominator. ^([2])p-value was calculated using the chi-square Test forcomparison between two treatment groups.

Table 58 summarizes the time points of wound recurrence. Woundrecurrence within 2 weeks was observed in 3 subjects (Subject 704,Subject 907, Subject 1002) in Granexin™ Gel plus SoC group and 2subjects (Subject 208, Subject 902) in SoC group.

TABLE 58 Timepoints of Wound recurrence Subject Visit Wound Visit WoundVisit Wound Number Treatment Group Healed recurred Healed Again 206Granexin ™ Gel Visit 9 Visit 13 — plus SOC 504 Granexin ™ Gel Visit 8Visit 18 — plus SOC 704 Granexin ™ Gel Visit 10 Visit 11 — plus SOC 907Granexin ™ Gel Visit 15 Visit 16 Visit 17 plus SOC 1002 Granexin ™ GelVisit 7 Visit 8 Visit 12 plus SOC 208 SOC Visit 10 Visit 11 Visit 16 902SOC Visit 8 Visit 9 Visit 10 1003 SOC Visit 9 Visit 16 — 1103 SOC Visit10 Visit 16 Visit 17 1107 SOC Visit 11 Visit 13 —

The intensity of pain experienced by the subjects was recorded on aVisual Analogue Scale of 1 to 10 where 1 indicated “no pain” and 10indicated “extreme pain” at all visits. The summary of subject selfassessment of intensity of pain at all visits from Baseline to Week 12for the ITT population is presented in and Table 59.

Table 59 summarizes the analysis of subject self assessment of intensityof pain at Week 12 for the ITT population. The intensity of pain was notstatistically significantly higher in subjects treated with Granexin™Gel plus SoC as compared to those treated with SoC only(p-value=0.8393).

TABLE 59 Analysis of Subject Self Assessment of Intensity of Pain atWeek 12 ITT Population (N = 92) Treatment Group Granexin ™ Gel plus SoCgroup SoC group Statistics (N = 46) (N = 46) Intensity of pain at Week12 n 39 38 Mean 0.46 0.34 SD 1.072 0.708 Median 0.00 0.00 Range(Min:Max) (0.0:5.0) (0.0:3.0) Missing 0 1 p-value ^([1]) 0.8393 Note:^([1]) p-value is evaluated by using non-parametric WilcoxonMann-Whitney U test between treatment group.

Table 60 summarizes the analysis of subject self assessment of intensityof pain at Week 12 for the mITT population. The intensity of pain wasnot statistically significantly higher in subjects treated withGranexin™ Gel plus SoC as compared to those treated with SoC only(p-value=0.1663).

TABLE 60 Analysis of Subject Self Assessment of Intensity of Pain atWeek 12 mITT Population (N = 77) Treatment Group Granexin ™ Gel plus SoCgroup SoC group Statistics (N = 41) (N = 36) Intensity of pain at Week12 n 34 29 Mean 0.18 0.45 SD 0.387 0.783 Median 0.00 0.00 Range(Min:Max) (0.0:1.0) (0.0:3.0) Missing 0 1 p-value ^([1]) 0.1663Treatment Group Granexin ™ Gel plus SoC group SoC group Statistics (N =46) (N = 46) Intensity of pain at Week 12 N 34 27 Mean 0.18 0.48 SD0.387 0.802 Median 0.00 0.00 Range (Min:Max) (0.0:1.0) (0.0:3.0)Treatment Group Granexin ™ Gel plus SoC group SoC group Statistics (N =41) (N = 36) Missing 0 1 p-value ^([1]) 0.1195 Note: ^([1]) p-value isevaluated by using non-parametric Wilcoxon Mann-Whitney U test betweentreatment group.

Wound Tracing were done for all subjects from Screening till Visit 15 ortill the time the wound had healed. Qualitative Assessment of the woundsite were done at the site at every visit for the presence ofgranulation, necrotic tissue, exudate levels, wound odor, and eschar aslisted in the CRF.

The non-parametric Wilcoxon Mann-Whitney U test was used to analyze meanpercent reduction of wound area from Baseline to Week 12 in both thetreatment groups since the data did not follow a normal distribution, asconfirmed by a Shapiro-Wilk p-value of <0.0001 and a non-linear Q-Qplot. The addition of ranks of both groups obtained after sorting thedata of wound size reduction in ascending order showed that in Granexin™Gel plus SoC subjects, the actual sum of scores was higher than theexpected sum of scores (ITT: actual: 1210.00, expected: 1071.00; mITT:actual: 1136.00, expected: 990.00; PP: actual: 1123.00, expected:973.50). In SoC group, the actual sum of scores was lower than theexpected sum of the scores (ITT: actual: 743.00, expected: 882.00; mITT:actual: 634.00, expected: 780.00; PP: actual: 588.00; expected: 737.50).Therefore, subjects in Granexin™ Gel plus SoC group had a statisticallysignificantly higher percent wound closure from Baseline to Week 12, ascompared to subjects in SoC group (ITT p-value=0.0238; mITTp-value=0.0105; PP p-value=0.0073).

Non-parametric analysis for secondary endpoint showed that the meanpercent reduction of wound area from Baseline to Week 4 in Granexin™ Gelplus SoC group was statistically significantly higher, as compared tosubjects in SoC group (ITT p-value=0.0006; mITT p-value=0.0001; PPp-value<0.0001).

Time to complete (100%) wound closure by Week 12 was statisticallysignificantly lesser in Granexin™ Gel plus SoC group, as compared to SoCgroup (ITT p-value=0.0006; mITT p-value<0.0001; PP p-value<0.0001). InGranexin™ Gel plus SoC group, 60.9% in the ITT population, 68.3%subjects in mITT population, and 80.0% subjects in the PP population had100% wound closure by Week 12 as compared to 32.6% in the ITTpopulation, 36.1% subjects in mITT population, and 36.4% in the PPpopulation in the SoC group. The median duration of 100% wound closurewas 6 weeks in ITT, mITT, and PP population in the Granexin™ Gel plusSoC group. In the SoC group, the median duration of time to completewound closure was about 12.14 weeks in the ITT population and notachieved in the mITT and PP population.

The time taken to achieve 50% wound closure by Week 12 was statisticallysignificantly lower in Granexin™ Gel plus SoC group as compared to SoCgroup subjects (ITT p-value=0.0002, mITT p-value<0.0001, PPp-value<0.0001). In Granexin™ Gel plus SoC group, 73.9% subjects in theITT, 80.5% subjects in mITT population, and 91.4% subjects in the PPpopulation had 50% wound closure and in SoC group, 54.3% subjects inITT, 63.9% subjects in mITT population, and 66.7% in PP population had50% wound closure. In ITT, mITT, and PP population, the median durationof 50% wound closure was 2.86 weeks in subjects treated with Granexin™Gel plus SoC. Subjects treated with SoC alone had median duration of6.86 weeks in the ITT population and 8 weeks in the both the mITT and PPpopulation.

In Cox Proportional Hazard Regression Analysis for time to 100% woundclosure covariates such as treatment group showed a significantassociation with time to completion of wound closure (ITTp-value=0.0406; mITT p-value=0.0139, PP p-value=0.0077) and theGranexin™ Gel plus SoC group subjects had ITT: 2.281; mITTp-value=2.776; PP: 3.150 times more chances of 100% wound closure thanthe SoC group subjects. Wound duration, wound size, baseline wounddepth, and BMI were not significant factors affecting wound closure.Treatment group and wound size showed a significant association withtime to completion of 50% wound closure (ITT: treatment groupp-value=0.0143; wound size p-value=0.0162; mITT: treatment groupp-value=0.0067; wound size p-value=0.0346; PP: treatment groupp-value=0.0059; wound size p-value=0.0442) and the Granexin™ Gel plusSoC group subjects had ITT: 2.247; mITT: 2.501 times; PP: 2.61 timesmore chances of wound closure than the SoC group subjects. Woundduration, baseline wound depth, and BMI were not significant factorsaffecting 50% wound closure.

The categorical analysis of incidence of 100% wound closure at Week 12was done using the Cochran-Mantel-Haenszel analysis with Breslow-Daytest and the Chi-square test. The Chi-square test performed by combiningthe data from all the study centers showed that the number of responderswith 100% wound closure in Granexin™ Gel plus SoC group (ITT: 56.5%;mITT: 63.4%; PP: 74.3%) was statistically significantly higher than inSoC group (ITT: 28.3%; mITT: 30.6%; PP: 30.3%) (ITT: p-value=0.0061;mITT p-value=0.0040; PP: p-value=0.0003).

The number of responders with 50% wound closure in Granexin™ Gel plusSoC group were 63% in ITT, 68.3% in mITT population as compared to 43.5%in ITT, 50% in mITT population in the SoC group which was notstatistically significant (ITT p-value=0.0600; mITT p-value=0.1025). Thenumber of responders with 50% wound closure in Granexin™ Gel plus SoCgroup in the PP population were 80% as compared to 51.5% in the SoCgroup which was statistically significantly higher (p-value=0.0131).

Hence, in the PP population, the frequency of 100% and 50% wound closure(epithelialization) was significantly higher in wounds treated withGranexin™ Gel plus SoC s than in SoC group subjects.

The intensity of pain experienced by the subjects was recorded on aVisual Analogue Scale of 1 to 10 where 1 indicated “no pain” and 10indicated “extreme pain”. There was no statistically significantdifference in subject self assessment of intensity of pain at the end oftreatment at Week 12 between the 2 treatment groups (ITT p-value=0.8393;mITT p-value=0.1663, PP p-value=0.1195, Wilcoxon Mann-Whitney U test).

Brief Summary of Adverse Events

Overall, 28 subjects reported 40 AEs of which 24 AEs were reported bysubjects in Granexin™ Gel plus SoC group; 16 AEs were reported in theSoC group. Out of 24 AEs, 21 (87.5%) in Granexin™ Gel plus SoC group andout of 16 AEs, 12 (75%) AEs in SoC group were mild. Three (12.5%) eventsin Granexin™ Gel plus SoC group and 3 (18.8%) events in SoC group weremoderate; and 1 (6.3%) event in SoC group was severe. None of the AEs ineither Granexin™ Gel plus SoC group or SoC group were related to thestudy drug or study treatment. Of the 40 events, 35 (87.5%) recoveredand 1 (2.5%) event recovered with sequelae, 3 (7.5%) AEs were ongoingwhen the subject completed the study. No action was taken against 39(97.5%) AEs. None of the subjects withdrew due to an AE and 1 (1.1%)subject in SoC group discontinued the study treatment due to death.

A total of 2 (2.2%) SAEs were reported in SoC group one of which wasdeath due to MI and the other was Deep Vein Thrombosis. Both the SAEswere not related to the study treatment. No SAEs were reported insubjects in Granexin™ Gel plus SoC group (Table 61).

TABLE 61 Overview of Adverse Events-Safety Population (N = 92) TreatmentGroup Granexin ™ Gel plus SoC SoC Overall Statistics (N = 46) (N = 46)(N = 92) Total number of AEs 24 16 40 reported Subjects reporting any 14(30.4%) 14 (30.4%) 28 (30.4%) AEs^([1]) Subjects reporting 1 8 (17.4%)12 (26.1%) 20 (21.7%) AE Subjects reporting >1 6 (13.0%) 2 (4.3%) 8(8.7%) AE Subjects Reporting 32 (69.6%) 32 (69.6%) 64 (69.6%) NoAEs^([1]) Number of AEs with severity of:^([2]) Mild 21 (87.5%) 12(75.0%) 33 (82.5%) Moderate 3 (12.5%) 3 (18.8%) 6 (15.0%) Severe 0(0.0%) 1 (6.3%) 1 (2.5%) Number of AEs with relationship of:^([2]) NotRelated 24 (100.0%) 16 (100.0%) 40 (100.0%) Unlikely 0 (0.0%) 0 (0.0%) 0(0.0%) Possible 0 (0.0%) 0 (0.0%) 0 (0.0%) Probable 0 (0.0%) 0 (0.0%) 0(0.0%) Definite 0 (0.0%) 0 (0.0%) 0 (0.0%) Number of AEs byoutcome:^([2]) Recovered 22 (91.7%) 13 (81.3%) 35 (87.5%) Recovered with0 (0.0%) 1 (6.3%) 1 (2.5%) sequelae On-going when 2 (8.3%) 1 (6.3%) 3(7.5%) subject completed the study Death 0 (0.0%) 1 (6.3%) 1 (2.5%)Unknown 0 (0.0%) 0 (0.0%) 0 (0.0%) Number of AEs by action taken:^([2])None 24 (100.0%) 15 (93.8%) 39 (97.5%) Discontinued 0 (0.0%) 1 (6.3%) 1(2.5%) study drug Subjects reporting 0 (0.0%) 0 (0.0%) 0 (0.0%) AEsleading to withdrawal^([1]) Subjects reporting 0 (0.0%) 2 (4.3%) 2(2.2%) SAEs^([1]) Subjects reporting 0 (0.0%) 1 (2.2%) 1 (1.1%) deathNote: ^([1])Percentage was calculated by taking respective column headergroup count as denominator. ^([2])Percentage was calculated by takingcount of ‘Total Number of AEs Reported’ in corresponding treatment groupas denominator.

Display of Adverse Events

The AEs by MedDRA system organ class and preferred term are summarizedin Table 62.

There was no statistically significant difference in the number ofsubjects with at least 1 AE between the treatment groups(p-value≧0.9999). More than 5% safety population reported venous ulcerpain (10 [10.9%]) and wound complication (12 [13.0%] subjects).

TABLE 62 Summary of Adverse Events by MedDRA System Organ Class andPreferred Term Safety Population (N = 92) Treatment Group n (%)^([1])Granexin ™ System Organ Class/ Gel plus SoC SoC Overall Preferred Term(n = 46) (n = 46) (N = 92) p-value ^([2]) Total number of subjects withat 14 (30.4%) 14 (30.4%) 28 (30.4%) ≧0.9999 least one AE Total number ofAEs reported 24 16 40 Cardiac disorders 0 (0.0%) 1 (2.2%) 1 (1.1%)Myocardial infarction^([3]) 0 (0.0%)[0] 1 (2.2%)[1] 1 (1.1%)[1] Generaldisorders and 0 (0.0%) 2 (4.3%) 2 (2.2%) administration site conditionsPyrexia 0 (0.0%)[0] 2 (4.3%)[2] 2 (2.2%)[2] Infections and infestations2 (4.3%) 1 (2.2%) 3 (3.3%) Wound infection 2 (4.3%)[2] 1 (2.2%)[2] 3(3.3%)[4] Injury, poisoning and procedural 2 (4.3%) 4 (8.7%) 6 (6.5%)complications Blister 1 (2.2%)[1] 0 (0.0%)[0] 1 (1.1%)[1] Woundcomplication 7 (15.2%)[11] 5 (10.9%)[5] 12 (13.0%)[16] Respiratory,thoracic and 2 (4.3%) 0 (0.0%) 2 (2.2%) mediastinal disorders Cough 1(2.2%)[1] 0 (0.0%)[0] 1 (1.1%)[1] Pneumonitis 1 (2.2%)[1] 0 (0.0%)[0] 1(1.1%)[1] Skin and subcutaneous tissue 7 (15.2%) 4 (8.7%) 11 (12.0%)disorders Venous ulcer pain 6 (13.0%)[6] 4 (8.7%)[4] 10 (10.9%)[10]Dermatitis allergic 1 (2.2%)[1] 0 (0.0%)[0] 1 (1.1%)[1] Pruritus 1(2.2%)[1] 0 (0.0%)[0] 1 (1.1%)[1] Vascular disorders 0 (0.0%) 1 (2.2%) 1(1.1%) Bleeding varicose vein 0 (0.0%)[0] 1 (2.2%)[1] 1 (1.1%)[1] Deepvein thrombosis 0 (0.0%)[0] 1 (2.2%)[1] 1 (1.1%)[1] Note:^([1])Percentage was calculated by taking respective column header groupcount as denominator. ^([2]) p-value was calculated by comparing twotreatment group using Chi-square test. ^([3])Myocardial infarction ledto death of the subject.

Of the 40 AEs reported in this study, one severe event of MI wasreported by a subject in the SoC group None of the AEs (Granexin™ Gelplus SoC group: 24 [100%]; SoC group: 16 [100.0%] had any definite,probable, or possible relationship to Granexin™ Gel or the studytreatment. One subject (Subject 1115) in SoC group was discontinued fromthe study treatment due to death. No other subjects were withdrawn ordiscontinued from the study due to an AE. Out of 40 AEs, 35 (87.5%)recovered, 1 (2.5%) AE (venous ulcer pain) in SoC group (Subject 102)recovered with sequelae, 3 (3.3%) AEs of wound complication (Granexin™Gel plus SoC group: 2 subjects [Subject 702, 916]; SoC group: 1 subject[Subject 1113]) were on-going when the subjects completed the study, and1 subject (Subject 1115, SoC group) died due to MI. One subject (Subject1115, SoC group) died due to MI.

At Screening visit, of the 9 female subjects in the study, 2 weresurgically sterilized and 4 were post menopausal. Pregnancy test wasdone for 3 (3.3%) subjects and doppler waveform analysis for all 92subjects. The mean Doppler waveform reading for Granexin™ Gel plus SoCgroup subjects was 11.60 cm/s and 9.57 cm/s for SoC group subjects.X-ray was taken for 86 (93.5%) subjects where X-ray finding was normalfor 81 (88.0%) and abnormal for 5 (5.4%) subjects, which was notclinically significant. None of the target wounds were infected with βhemolytic Streptococcus culture. The eGFR for males in Granexin™ Gelplus SoC group was 95.18 mL/min and 88.50 mL/min in SoC group subjects.However, the eGFR for female and ABPI were comparable between thetreatment groups.

None of the subjects in the study had clinically significanthematological parameters at Screening visit or Week 12. Two subjects inGranexin™ Gel plus SoC group had clinically significant HbA1c atScreening visit but none of the subjects had clinically significantHbA1c at 12 weeks. None of the subjects had any clinically significantcreatinine, uric acid, BUN, potassium, sodium, chloride, bicarbonate,albumin, AST, ALT, and total cholesterol at Screening visit or at Week12.

Immunogenicity Testing: Using universal precautions recommended forbiological samples, the blood samples were collected in yellow top gelvaccutainers. They were properly labeled with date and time ofcollection, site number, visit number, screening and randomizationnumber, and the sample type. Using acceptable venipuncture technique 7mL of whole blood was collected and allowed to stand for 15 min for theclot to form. Serum was separated by centrifuging the blood samples at3500 rpm. Approximately 2 mL of the separated serum was pipetted outinto two aliquots and refrigerated at 2-6° C. till shipped. These tubeswere labeled with date and time of collection, site number, visitnumber, screening and randomization number, and the sample type. Thesesamples were shipped to a central storage facility (MetropolisLaboratory, Mumbai) under appropriate temperature conditions with dryice or frozen cold packs.

The samples were stored at −20° C. at Metropolis and shipped in batchesto WuXi Laboratories at Philadelphia, Pa., under appropriate temperatureconditions with serum sample shipment forms.

Anti-ACT1 antibodies were not detected in the serum in any of thesubjects at Screening and Week 12.

The systolic and diastolic blood pressure, heart rate, respiratory rate,and body temperature were comparable between the treatment groups atScreening visit and at Week 12.

In the overall safety population, the ECG was normal in 81 (88.0%)subjects. Changes in ECG impression reported in the remaining subjectswere not clinically significant at the Screening visit (End-of-TextTable 14.3.6)

Overall, 28 (30.4%) subjects reported 40 AEs of which 24 AEs werereported by subjects in Granexin™ Gel plus SoC group; 16 AEs werereported in the SoC group. Out of 24 AEs, 21 (87.5%) in Granexin™ Gelplus SoC group and out of 16 AEs, 12 (75%) AEs in SoC group were mild.Three (12.5%) events in Granexin™ Gel plus SoC group and 3 (18.8%)events in SoC group were moderate; and 1 (6.3%) event in SoC group wassevere. None of the AEs in either Granexin™ Gel plus SoC group or SoCgroup were related to the study drug or study treatment. Of the 40events, 35 (87.5%) recovered and 1 (2.5%) event recovered with sequelae.None of the subjects withdrew due to an AE and 1 (1.1%) subject in SoCgroup discontinued the study treatment due to death.

A total of 2 (2.2%) SAEs were reported in SoC group one of which wasdeath due to Myocardial Infarction and the other was Deep VeinThrombosis. Both the SAEs were not related to the study treatment. NoSAEs were reported by subjects in Granexin™ Gel plus SoC group Overall,there was no significant difference in the number of subjects reportingAEs between the 2 treatment groups (Granexin™ Gel plus SoC group, 14;SoC group, 14; p-value≧0.9999).

At Screening visit, no clinically significant abnormalities wereobserved in any of the treatment groups. The vital signs and ECG werenormal and comparable between both the treatment groups. Anti-ACT1peptide antibodies were not detected in any of the subjects in bothScreening and EOS serum samples.

This was a double blind, randomized, prospective, parallel group,multi-center Phase II study conducted at 10 centers in India to evaluatethe efficacy and safety of Granexin™ Gel in the treatment of VLU. Thetotal duration of the study was 11 months, including 5 months enrollmentperiod and 6 months for study procedures. The study duration forindividual subjects was 6 months which included 18 visits. A total of101 subjects with VLU were screened for this study, 5 were found to bescreen failures, and 4 subjects did not initiate participation in thestudy. A total of 92 subjects enrolled in the study had mean woundduration of 68.4 weeks at Screening and mean wound area of 353.64 mm² atBaseline. These subjects were randomized to receive Granexin™ Gel plusSoC or SoC alone for the treatment of VLU. Of these, 70 completed thestudy and 22 subjects did not complete the study due to consentwithdrawal, loss to follow-up, non-compliance, or SAE.

Of 92 enrolled subjects, 15 subjects met exclusion criterion number 1 ordid not have adequate evaluable wound photographic data postrandomization but were considered in the Safety and ITT population.These subjects were excluded from the mITT and PP populations. A totalof 83 (90.2%) subjects were males and 9 (9.8%) were females. The meanage of the study population was 49.8 years, mean weight was 70.47 kg,mean height was 168.35 cms, and mean BMI was 24.79 kg/m².

The primary efficacy endpoint of the study was to evaluate mean percentwound closure from Baseline (Visit 2) to Week 12 (Visit 15). Thenon-parametric Wilcoxon Mann-Whitney U test was used to analyze meanpercent reduction of wound area from Baseline to Week 12 in both thetreatment groups since the data did not follow a normal distribution, asconfirmed by a Shapiro-Wilk p-value<0.0001 and a non-linear Q-Q plot.Subjects treated with Granexin™ Gel plus SoC showed statisticallysignificantly higher percent wound closure from Baseline to Week 12compared to subjects treated with SoC alone (ITT p-value=0.0238; mITTp-value=0.0105; PP p-value=0.0073), suggesting that at Week 12, woundstreated with Granexin™ Gel plus SoC had a significant reduction in sizeas compared to wounds treated with SoC alone.

Mean percent wound closure at Week 4, time to complete (100% and 50%)wound closure, incidence of complete wound closure, and subject selfassessment of pain were also analyzed. At Week 4, the wound area percentof reduction in Granexin™ Gel plus SoC group was statisticallysignificantly higher than in SoC group (ITT p-value=0.0006; mITTp-value=0.0001; PP p-value<0.0001 Wilcoxon Mann-Whitney U test).

Subjects treated with Granexin™ Gel plus SoC achieved 100% wound closurein median duration of 6 weeks in ITT, mITT, and PP population which wassignificantly faster compared to the SoC group in which the medianduration was 12.14 weeks in the ITT population and not achieved in bothmITT and PP population during the 12 weeks of efficacy assessments (ITTp-value=0.0006; mITT p-value=0.0001; PP p-value<0.0001; log-rank test).

Subjects treated with Granexin™ Gel plus SoC achieved 50% wound closurein 2.86 weeks in both ITT and PP population while subjects treated withSoC alone achieved it in 6.86 weeks in ITT population and 8 weeks inboth mITT and PP population, respectively (ITT p-value=0.0002, mITTp-value<0.0001, PP p-value<0.0001). The categorical analysis ofincidence of 100% and 50% wound closure at Week 12 was conducted and thesubjects who had 100% wound closure were considered as responders.Overall in the ITT, mITT, and PP populations, 56.5%, 63.4%, and 74.3% ofthe subjects in Granexin™ Gel plus SoC group, respectively, responded totreatment with 100% wound closure as compared to 30.3% of the subjectsin the SoC group. The number of responders were significantly higher inGranexin™ Gel plus SoC group than in the SoC group (ITT: p-value=0.0061;mITT p-value=0.0040; PP: p-value=0.0003; Chi-square analysis).Similarly, in the PP population, 80.0% of the subjects in Granexin™ Gelplus SoC group responded to treatment and achieved 50% wound closure ascompared to 51.5% of the subjects in the SoC group. The number ofresponders with 50% wound closure were significantly higher in Granexin™Gel plus SoC group than in the SoC group (p-value=0.0131; Chi-squareanalysis), indicating that the wounds treated with Granexin™ Gel plusSoC have higher incidences of 100% and 50% wound closure(epithelialization) than wounds treated with SoC alone.

Cox Proportional Hazard Regression Model analysis was conducted toevaluate if one or more covariates such as treatment group, woundduration, baseline wound depth, and BMI etc. were associated with thetime to complete 100% or 50% wound closure. It showed that treatmentgroup was a significant factor affecting wound closure and Granexin™ Gelplus SoC group subjects had ITT: 2.281; mITT: 2.776; PP: 3.150 timesmore chances of 100% wound closure and ITT: 2.247; mITT: 2.501; PP: 2.61times more chances of 50% wound closure than the SoC group subjects.

At every visit, self assessment of intensity of pain score was completedby each subject and the scores were given in comparison to the lastvisit. Therefore significant change in the pain scores was not observedand there was no statistically significant difference in intensity ofpain as assessed by the subject at Week 12 between the 2 treatmentgroups.

The findings from the present study demonstrated a good safety profilewith Granexin™ Gel. Overall, 28 subjects reported 40 AEs of which 24 AEswere reported by subjects in Granexin™ Gel plus SoC group; 16 AEs werereported in the SOC group. The only 2 SAEs reported, were in the SoCgroup. One SAE was Myocardial infarction which resulted in death, andthe other SAE was Deep vein thrombosis. Of the 40 AEs, 35 (87.5%)recovered. None of the AEs were related to Granexin™ Gel. None of thesubjects were withdrawn from the study due to an AE.

No clinically significant abnormalities in the laboratory parameterswere observed in either of the treatment groups. The vital signs and ECGwere normal and comparable between both the treatment groups. Anti-ACT1peptide antibodies were not detected in the serum samples collected atScreening or at Week 12.

In conclusion, Granexin™ Gel along with SoC has shown to acceleratewound healing in subjects with VLU, making it an efficacious, safe, andwell tolerated therapeutic option in the treatment of VLU.

Unless defined otherwise, all technical and scientific terms herein havethe same meaning as commonly understood by one of ordinary skill in theart to which this invention belongs. Although any methods and materials,similar or equivalent to those described herein, can be used in thepractice or testing of the present invention, the preferred methods andmaterials are described herein. All publications cited herein areincorporated herein by reference for the purpose of disclosing anddescribing specific aspects of the invention for which the publicationis cited.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth and as follows in the scope ofthe appended claims.

TABLE 9 Wound Healing Gel Qualitative and Quantitative CompositionConcentration Grams/ Ingredients Grade Function (% w/w) kg Batch Peptide328967 — Active 0.0072; 0.018 0.072; 0.18 (ACT1) 0.036; 0.072 0.36; 0.72Methylparaben NF Preserva- 0.17 1.7 tive Propylparaben NF Preserva- 0.020.2 tive Glycerin USP Solvent 5 50 Sodium Phosphate USP Buffer 0.2632.63 Monobasic Agent Sodium Phosphate USP Buffer 0.044 0.44 DibasicAgent Propylene Glycol USP Solvent 3 30 Edetate Disodium USP Chelating0.05 0.5 (EDTA) Agent D-Mannitol USP Stabilizer 0.05 0.5 Hydroxyethyl-NF Gelling 1.25 12.5 cellulose, 250HHX Agent Purified Water, USP Solvent1 1 kg qsad

1-32. (canceled)
 33. A topical formulation comprising at least one alphaconnexin polypeptide and hydroxyethylcellulose gel, wherein thehydroxyethylcellulose gel stabilizes the alpha connexin polypeptide. 34.The topical formulation of claim 33, wherein the hydroxyethylcellulosegel stabilizes the alpha connexin polypeptide so that after 3 months ofstorage at 5° C. at least 98% of the alpha connexin polypeptide isdetectable by analytical methods.
 35. The topical formulation of claim33, wherein the hydroxyethylcellulose gel stabilizes the alpha connexinpolypeptide so that after 3 months of storage at 5° C. at least 95% ofthe alpha connexin polypeptide is detectable by analytical methods. 36.The topical formulation of claim 33, wherein the hydroxyethylcelluloseis present at a concentration of about 1.25% (w/w).
 37. The topicalformulation of claim 33, wherein the at least one alpha connexinpolypeptide is present at a concentration of between about 0.005% (w/w)and about 1.00% (w/w).
 38. The topical formulation of claim 33, furthercomprising a buffering agent.
 39. The topical formulation of claim 38,wherein the buffering agent maintains the pH of the topical formulationbetween about 5 and about
 7. 40. The topical formulation of claim 38,wherein the buffering agent is a phosphate buffer.
 41. The topicalformulation of claim 33, wherein the at least one alpha connexinpolypeptide comprises the carboxy-terminal most 4 to 30 contiguous aminoacids of an alpha connexin protein or conservative variant thereof. 42.The topical formulation of claim 41, wherein the conservative variantcomprises up to two conservative substitutions in the amino acidsequence of the alpha connexin polypeptide.
 43. The topical formulationof claim 33, wherein said at least one alpha connexin polypeptide islinked at its amino terminus to a cellular internalization transporter.44. The topical formulation of claim 43, wherein the cellularinternalization transporter is an antennapedia sequence.
 45. The topicalformulation of claim 33, wherein the at least one alpha connexinpolypeptide comprises the amino acid sequence of SEQ ID NO:
 9. 46. Thetopical formulation of claim 33, wherein the alpha connexin polypeptideis connexin 37, connexin 40, connexin 43, or connexin
 45. 47. A methodof wound treatment comprising administering the topical formulation ofclaim 33 to a subject in need thereof.
 48. The method of claim 47,wherein the wound is an acute surgical wound.
 48. The method of claim47, wherein the wound is a chronic, non-infected, full-thickness lowerextremity ulcer.
 49. A method of manufacturing a topical formulationcomprising: a) mixing propylene glycol, glycerin, methylparaben andpropylparaben until the parabens are completely dissolved; b) separatelymixing purified water, EDTA, monobasic sodium phosphate, dibasic sodiumphosphate and D-mannitol until a clear solution is obtained; c) addingthe solution from a) to the solution from b), rinsing the container ofthe solution from a) with purified water, adding the rinse to thecombined solutions, and mixing until the combined solutions are visuallyhomogeneous; d) with homogenization mixing, adding hydroxyethylcellulose into the combined solutions of c) and mixing until the polymeris fully dispersed; e) separately mixing purified water with an alphaconnexin polypeptide until the peptide is completely dissolved; f)adding the solution from e) to the solution of d), rinsing the containerof the solution from e) with purified water, adding the rinse to thecombined solutions, and mixing until the combined solution arehomogeneous.